No effect was observed on performance in interaction of organic acids together and with PHY group (p > 0.05). In duodenum CA increased the villus height and width (p ˂ 0.05) and PHY enzyme increased villus width (p ˂ 0.05) and decreased crypt depth (p ˂ 0.05). On the other hand, CA along with PHY significantly decreased crypt depth (p ˂ 0.05). In jejunum PHY alone and in combination with vinegar increased the goblet cells numbers (p ˂ 0.05), whereas vinegar significantly increased the goblet cells numbers in ileum (p ˂ 0.05). The muscular thickness in duodenum, jejunum, and ileum was not aﬀected among diﬀerent treatment groups. The results showed that supplementation of organic acids and phytase together in this experiment, with no negative effects on each other, improved their effects on some parameters.The present experiment was conducted to evaluate the effects of organic acids and phytase enzyme supplementation on performance and intestinal histomorphology of broilers. The experiment was done in a factorial arrangement 2 × 2 × 2 based on completely randomized design with eight treatments, five replicates with 12 chicks in each until 42 days of age. Diets included natural vinegar (0 and 2%), citric acid  and phytase enzyme . One bird from each treatment replicate was randomly selected and slaughtered to evaluate the small intestinal morphology on 42 days of age. Analysis of results showed that vinegar increased feed consumption and body weight gain in total experimental period ( Organic acids are widespread in plant and animal tissues. It was reported that propionic, formic, citric, lactic and ascorbic acids increase nutrient digestibility without harmful affecting performance.4 Organic acids inhibit the growth of intestinal bacteria leading to reduce metabolic needs by increasing the availability of nutrients to the host.10 In most cereal grains and oilseeds the phytate phosphorus (PP) is the main storage form of phosphorus and represents 50.00 to 85.00% of the total phosphorus.11 Phytate phosphorus is not available to the poultry because they do not contain sufficient amounts of intrinsic phytase (PHY) required to hydrolyze the PP.12 Since there is no natural PHY in the gastrointestinal tract of the poultry, supplementation of diets with exogenous PHY has been proven to enhance the digestibility of phytate phosphorus.13 Citric acid (CA) can improve the efficacy of PHY because it can chelate multivalent cations.14 Acidiﬁcation with various organic acids has been reported to reduce the production of toxic components by the bacteria and colonization of pathogens on the intestinal wall, thus preventing damage to epithelial cells.15The intestinal microflora of broilers have beneficial effects on the nutrients, making them more available from the intestinal tract by affecting apparent digestibility of protein, nitrogen, and fat, depending on the composition of the diet.There is limited information about the effects of organic acids and PHY supplementation on the performance and intestinal histomorphology of the broiler chickens and the objectives of this study was to evaluate them.16 Treatments included: 1) basal diet, 2) basal diet + 2.00% natural grape vinegar, 3) basal diet + 1.00% citric acid, 4) basal diet + 500 FTU kg-1 PHY, 5) basal diet + 2.00% natural grape vinegar + 1.00% CA, 6) basal diet + 2.00% natural grape vinegar + 500 FTU kg-1 PHY, 7) basal diet + 1.00% citric acid + 500 FTU kg-1 PHY, 8) basal diet + 2.00% natural grape vinegar + 1.00% CA + 500 FTU kg-1 PHY. Ingredient composition and the calculated nutrients composition are given in Birds and dietary treatments. One-day-old male Ross 308 broiler chickens (n = 480) were individually weighed and pen averages were adjusted so that no significant difference among pens was realized. The chickens were allocated to 40 pens in a 2 × 2 × 2 factorial arrangement based on completely randomized design with eight treatments, five replicates and 12 chickens in each. Wood shavings were used as litter. A basal diet was formulated with wheat-corn-soybean meal according to the Ross 308 recommendations for all nutrients.nd day of age), five broiler chickens of similar body weight with the group average were selected from each group and slaughtered by severing a jugular vein. Samples of duodenum, jejunum and ileum were obtained from the slaughtered birds and approximately two cm of the middle portions of the duodenum, jejunum and ileum were excised and fixed in 10% buffered formalin for one week. Tissues were dehydrated by immersing through a series of alcohols with increasing concentration (from 70% to absolute), infiltrated with xylene and embedded in paraffin.Measurements. The body weight gain of birds per replicate was recorded on the individual basis at weekly intervals. The cumulative feed consumption per replicate was also recorded on a weekly basis. Feed conversion ratio per replicate was worked out at weekly intervals by taking into consideration the weekly body weight gain and the feed consumption of respective replicate. For histomorphological assessment, at the end of the experiment . The blocks were properly trimmed and the sections of 7.00 µm thickness were cut. The tissue sections were stained by hematoxylin and eosin for measuring the villus height, villus width, crypt depth and muscular thickness. Acidic mucus containing goblet cells were identified using periodic acid shift (PAS). All of the specimens were studied by multiple magnifications (400× and 1000×).4Statistical analysis. Data were analyzed using the general linear models (GLM) procedure in SAS software  with the main effects of natural grape vinegar, CA, and PHY and their two- and three-way interactions included in the model. The differences among group means were verified statistically by analysis of variance using Tukey’s test at p ˂ 0.05. The following model statement was used:ijkl = μ + Pi + Cj + Sk + PC (ij) + PS (ik) + CS (jk) + PCS (ijk) + eijkl Yijkl is the response measured, μ is the overall mean, Pi is the effect of PHY, Cj is the effect of natural grape vinegar level, Sk is the effect of CA level, PC(ij) is the interaction between PHY and natural grape vinegar, PS(ik) is the interaction between PHY and CA, CS(jk) is the interaction between natural grape vinegar and CA, PCS(ijk) is the interaction among natural grape vinegar, CA acid, and PHY, and eijkl is the residual error.where, YPerformance. Effect of dietary treatments on growth performance, including body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR), are summarized in p ˂ 0.05) whereas there was not any notable effect on 0 to 14 days of age. The CA and PHY did not have any effect on BWG. The interaction of organic acids together and with PHY did not have any effect on performance.Histomorphology. The mean values regarding the histomorphological alterations in broilers fed organic acids and PHY are given in p ˂ 0.05). The PHY increased the villuswidth (p ˂ 0.05) and decreased the crypt depth (p ˂ 0.05) and in combination with CA decreased the crypt depth and increased villus height to crypt depth ratio significantly (p ˂ 0.05) while vinegar alone or in combination with PHY did not have any significant effect on characteristics of duodenum. In jejunum, PHY alone and in combination with vinegar increased the goblet cell numbers (p ˂ 0.05) whereas vinegar significantly increased the goblet cell numbers in ileum (p ˂ 0.05). The muscular thickness in the duodenum, jejunum and ileum was not aﬀected by diﬀerent treatment groups. Citric acid and PHY alone and together increased the ratio of villus height to crypt depth in duodenum (p ˂ 0.05).18 The organic acids may aﬀect the integrity of microbial cell membrane or cell macromolecules or interfere with the nutrient transport and energy metabolism causing the bactericidal effect. In this study, the possible improvement in performance by vinegar may be attributed to the nutrients in vinegar. Acetic acid is the main component of vinegar. Some other constituents include, anthocyanins (e.g. cyanidin-3- glucoside) flavonols , flavanols ,19 vitamins, mineral salts, amino acids and nonvolatile organic acids .20 Therefore, vinegar has more effects rather than citric acid on performance of broilers. Addition of vinegar to mash diet may lead to increase in palatability and improve mixing ration items, resulting in increased FI and consequently an increase in BWG. These results were in concordance with the reports of earlier researchers. Abdel-Fattah et al. 21 reported that the addition of dietary acetic acid improved live body weight of broiler chicks compared to those fed on unsupplemented diets. Kopecký et al. concluded that supplementation of citric acid caused decrease in total feed consumption;5 that was in agreement with the results of Afsharmanesh and Pourreza, who reported that the addition of citric acid to broiler diet improved feed efficiency. 22 Sacakli et al. found that feed consumption and feed conversion ratio were not affected by addition of PHY or organic acid alone or in combination to diet.6 In the same way, Houshmand et al.,23 and Hernandez et al., have also observed no significant effect of organic acid supple-mentation on growth performance of broilers.24 Adil et al. reported that supplementation of organic acids in broilers improved BWG when compared to the unsupplemented group.10 They reported that improved BWG is probably due to the beneﬁcial effects of organic acids on the gut ﬂora.10 Numerous factors, such as environment, farm rearing conditions, management practices, nutrition, organic acid type and concentration, composition of diet and bird characteristics  can affect the response of broilers to feed additives.8,25 Thus, effects of citric acid in the present study could be attributed to above mentioned factors. Results on growth-promoting effects of organic acids have been inconsistent.26 This inconsistency in results could be attributed to several factors. One of the most important factors is the buffering capacity of dietary feedstuffs. The beneficial effects of antimicrobial additives on growth performance would be apparent under poor hygienic conditions or when feeding poorly digestible diets.26 This study was carried out under good rearing conditions. Under good conditions, broilers did not need any feed additives for maximum growth performance.27 Kishi et al. reported that improved body weight gain is probably due to the beneﬁcial effects of organic acids on the gut ﬂora.28 Increased villus height provides a greater surface area for nutrients absorption and consequently, higher performance.28 On the contrary, reduction in villus height can reduce nutrients absorption due to the decrease in the intestinal surface area for absorption. Thus, reducing nutrients absorption decreases resistance to disease and lower growth performance. Increase in secretions of gastrointestinal tract are the negative consequence of deeper crypt and shorter villi.29 Pelicano et al. reported increased villus heights in duodenum and jejunum with most of the organic acidiﬁers which they attributed to the fact that organic acids reduce the growth of many pathogenic or nonpathogenic intestinal bacteria, decreasing the intestinal colonization and infectious processes, ultimately decreasing the inﬂammatory reactions at the intestinal mucosa, which increases the villus height and functions of secretion, digestion and absorption of nutrients by the mucosa.30 The reduction in the muscular thickness is helpful in improving the digestion and absorption of nutrients as reported by Teirlynck et al.31 The thickening of mucous layer on the intestinal mucosa contributes to the reduced digestive eﬃciency and nutrients absorption.31 Garcia et al. reported that broiler chickens fed on formic acid had the greater villus height and width, and crypt depth compared to the control group.26 Reportedly, reported that the duodenum villus height was significantly increased with the different levels of CA compared to the control.21,32 Moreover, the presence of other antimicrobial compounds, organic acid type and concentration, composition of diet and the experimental environment are factors that could affect response of birds to organic acids.8Height and width of villus could be considered as indicators for an active functioning of intestine.In conclusion, this study pointed out the importance of using organic acid as physiological additives to increase the growth performance and intestinal histomorphology of broilers through their physiological action in inducing the growth and activities of some endogenous mechanisms responsible for better performance. As well, under the condition of this study, no further benefits were achieved as a result of increasing the dietary organic acids and PHY levels. The reason of these results may be due to type and dose of organic acids and PHY in these studies. Further studies are needed to throw more light on developmental effects of those organic acids on the performance and intestinal histomorphology of broiler chickens.


Introduction: There are still uncertainties about the true nature of age related changes in topological properties of the brain functional network and its structural connectivity during various developmental stages. In this cross- sectional study, we investigated the effects of age and its relationship with regional nodal properties of the functional brain network and white matter integrity.Method: DTI and fMRI data were acquired from 458 healthy Chinese participants ranging from age 8 to 81 years. Tractography was conducted on the DTI data using FSL. Graph Theory analyses were conducted on the functional data yielding topological properties of the functional network using SPM and GRETNA toolbox. Two multiple regressions were performed to investigate the effects of age on nodal topological properties of the functional brain network and white matter integrity.Result: For the functional studies, we observed that regional nodal characteristics such as node betweenness were decreased while node degree and node efficiency was increased in relation to increasing age. Perversely, we observed that the relationship between nodal topological properties and fasciculus structures were primarily positive for nodal betweenness but negative for nodal degree and nodal efficiency. Decrease in functional nodal betweenness was primarily located in superior frontal lobe, right occipital lobe and the global hubs. These brain regions also had both direct and indirect anatomical relationships with the 14 fiber bundles. A linear age related decreases in the Fractional anisotropy (FA) value was found in the callosum forceps minor.Conclusion: These results suggests that age related differences were more pronounced in the functional than in structural measure indicating these measures do not have direct one-to-one mapping. Our study also indicates that the fiber bundles with longer fibers exhibited a more pronounced effect on the properties of functional network. Aging is considered as one of the greatest risk factors for neurodegenerative diseases where there is progressive loss of either structure or function of the neurons , which is a MRI modality based on principle of water diffusion measures  and characteristics path length (Lp)]; efficiency metrics ; and regional nodal properties   have examined the association of structural and functional connectivity of the cingulum bundle to probe the cognitive and motor performance, where they have suggested that only structural connectivity but not resting state functional connectivity was significantly associated with age. Since, the characterization on structural and functional connectivity is essential to determine the relationship between them. We, therefore have made an effort to ascertain the nature of functional connections by examining the strength of structural connections and vice-versa in a large sample of healthy subjects (n = 458) with an age range of 8–81 years. We hypothesize that changes in both white matter integrity and functional connectivity would be observed in certain networks, and these changes in nodal topological properties of the functional network would be associated with the changes in FA.Despite of many advances in neuroscience, there is still much to learn about the relationship between white matter integrity and nodal topological properties of structural and functional network. So far, Cao et al.  has emplDTI and fMRI data were acquired on 3T MRI systems from 458 healthy Chinese Han subjects  at West China Hospital of Sichuan University. The subjects were recruited via local advertising. Among these, 443 participants had fMRI data, 346 participants had DTI data, and 331 participants had both fMRI and DTI data , voxel size, 3.75 × 3.75 × 5 mm3; matrix, 64 × 64. Parameters of Siemens machine were: repetition time = 2,000 ms, echo time = 30 ms, field of view (FOV) = 24 × 24 cm2, flip angle = 90°, slice thickness = 5.0 mm (no gap), voxel size 3.75 × 3.75 × 5 mm3, matrix 64 × 64.The subjects underwent a resting-state fMRI scan in one of two 3T MRI systems [General Electric  or Siemens ]. Parameters of General Electric machine were; repetition time = 2,000 ms, echo time = 30 ms, field of view (FOV) = 24 × 24 cm2, resolution matrix 128 × 128, flip angle = 90°, slice thickness = 3.0 mm, A total of 672 slices were acquired for b values of b = 0 and b = 1,000 mm2/s, which were obtained by applying gradients along 15 non-collinear directions. Parameters of Siemens machine were; TR = 6,800 ms, TE = 93 ms, field of view (FOV) = 24 × 24 cm2, resolution matrix 128 × 128, flip angle = 90°, slice thickness = 3.0 mm. 42 slices were acquired for b = 0 and b = 1,000 mm2/s; these were obtained by applying gradients along 30 non-collinear directions.DTI data were also acquired from one of two 3T MRI systems. Parameters of General Electric machine were: TR = 10,000 ms, TE = 70.8 ms, field of view (FOV) = 24 × 24 cmPreprocessing contained both functional and structural preprocessing steps.http://www.fil.ion.ucl.ac.uk/spm) and GRETNA  ,” which implements both algorithms proposed by Hua and Zhang . Two multiple regressions were performed to investigate the effects of age on nodal topological properties of the functional brain network and white matter integrity with nodal topological properties and mean FA values as dependent variables while; age, gender, and education kept as independent variables. Additionally, multiple regression model was also used to explore the association between nodal topological properties and white matter integrity by using the value of each nodal topological property as an independent variable and mean FA value of each fiber bundle, maintaining age, gender, and education as dependent variables. Finally, two subgroup analyses were performed to investigate the interactive effect between gender and nodal topological properties or white matter integrity.P < 0.05).We performed linear regression to analyze the effect of age on the topological properties  were calculated for global efficiency , local efficiency , and the clustering coefficient . A linear age-related reduction in path length was also observed .Linear age-related changes in the node betweenness were revealed in 21 brain regions. Positive correlation between age and node degree and node efficiency was observed in bilateral middle and inferior frontal gyrus, bilateral anterior and median cingulated cortex, bilateral postcentral gyrus, superior parietal gyrus, bilateral superior temporal gyrus, middle temporal gyrus, bilateral caudate nucleus and left lenticular nucleus (Table p < 0.001)  Figure . HoweverAs observed in Supplementary Table In this study, we investigated age related changes in topological properties of brain functional network and structural connectivity. We observed that the nodes and topological properties that changed with increasing age were limited to specific regions, and FA values of most fiber bundles did not alter with increasing age, suggesting that age related changes are more pronounced in functional rather than structural connectivity measures indicating these measures do not have a direct one-to-one mapping. It was also noted that fiber bundles with longer fibers exhibited more pronounced effect on the properties of functional network rather than structural network.For regional characteristics of functional properties, we observed that age related decreases in node betweenness were primarily located in the superior frontal lobe, right occipital lobe and global hubs. Among 21 brain regions that had significant relationship with age, 11 global hubs identified crucial to efficient communication  age ranging from 18 to 85 indicated an anterioposterior gradient of age related decline in corpus callosum fibers, where a potential role of regional white matter damage  in influencing different cognitive performances in healthy subjects was noted. One of the reasons for different findings might be due to difference in sample size, where a larger sample size might have permitted retention of more paths in their study. In addition, larger number of sample subjects between the age group 20 and 40 in our study might also play its role in different findings between these two studies. Other reason includes the methodological differences in these studies where Voineskos and colleagues did not study all white matter tracts . The current study also indicates that the FA value of the most of the fiber bundles did not substantially change with age. However, a direct inference on anatomical connectivity differences cannot be based on this observation alone. The unchanged FA value might also be due to a balance created among alterations in fiber size, density, and myelination or fiber coherence. However, these differences in findings also suggest that white matter alterations are variable throughout the brain.Other finding of our study was that the FA values of most of the fiber bundles did not alter with increasing age except that of callosum forceps minor. This age-dependent decrease in FA in the callosum forceps minor was in accordance with findings from several recent studies on the age-related alterations in the white matter microstructure , local efficiency , and clustering coefficient   Figure . Small-w) Figure and have Several issues must be addressed while considering the results of our study. First, the population distribution of our study was not uniform. Proportions of subjects between 20 and 40 year was larger than that of other age groups. Second, we used a DTI-based streamline tractography approach (Mori et al., Current study indicates that, age related changes are more pronounced in the functional than in structural measure indicating these measures do not have a direct one-to-one mapping. Frontal regions are more vulnerable with advancing age and fiber bundles with longer fibers exhibited pronounced effect on the functional network properties. Future longitudinal studies would be useful to investigate the changes in the functional and anatomical neural networks that occur with normal aging.Study concept and design: SL and QG. Acquisition, analysis, or interpretation of data: HS and JiaL. Drafting of the manuscript: CS and JiaL. Critical revision of the manuscript for important intellectual content: SL and PL. Statistical analysis: HS, JieL, and WZ. Obtained funding: SL and QG. Adminstrative, technical, or material support: CS, YX, YZ, and LY. Study supervision: SL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GSTM1) gene in humans has been shown to be involved in xenobiotic metabolism and associated with bladder cancer. However, the evolution of this deletion has not been investigated.The common deletion of the glutathione S-transferase Mu 1  contains supplementary material, which is available to authorized users. For example, cytochrome P450 2D (CYP2D) gene family underwent frequent gene duplications, losses and gene conversions in primates with implications to drug metabolism variation in humans [CYP gene family members have been reported to evolve under non-neutral forces in humans, possibly as a response to variation in dietary intakes, such as salt consumption [CYP genes, NAT2 (N-acetyltransferase 2) has also been reported to be evolving under non-neutral conditions in non-human primates, as well as among human populations [UGT2B17 (uridine diphosphoglucuronosyltransferase) has been shown to adaptively increase in frequency in East-Asian populations [GST superfamily, GSTT1, was unusually old and showed signatures of balancing selection. The diverse evolutionary trajectories of metabolizing genes are often attributed to changes in dietary xenobiotic exposure during evolutionary time [Metabolizing genes, such as the members of the n humans , 21. Morn humans . Moreovesumption , 24. Simulations , 21, 25.ulations . Last buulations  found thary time , as wellary time .GSTM gene family varies in composition among primates with unknown functional consequences. For example, a locus-specific study has shown that the GSTM1 was not expressed as a functional gene in a cynomolgus macaque (Macaca fascicularis) [GSTM1 gene, may have been have been evolving under non-neutral conditions.The cularis) . Rapid ccularis) . In addicularis) . OverallGSTM1 gene has likely been facilitated by the architecture of the GSTM locus. The GSTM family members were generated by multiple segmental duplications, which are near identical segments of DNA larger than 1 kb [GSTM1 deletion has formed. Specifically, segmental duplications tend to cause gene duplication and deletion events by facilitating non-allelic homologous recombinations [GSTM locus, Uno et al. [GSTM1 in humans is a result of non-allelic homologous recombination event facilitated by two segmental duplications flanking this gene in the primate genomes [GSTM locus among primates and specifically the origins of the GSTM1 gene deletion in humans.The deletion of the han 1 kb . These sinations . Consequinations . In factinations –36. For o et al.  reportedGSTM1 deletion, we first conducted in silico comparative genomic analyses among primates . We confirmed the presence of the chimpanzee polymorphic deletion using Droplet Digital PCR  and read-depth methods in 4 chimpanzee samples used in a previous study [Next, we extensively investigated the presence of a deletion , 38  with each other and also with the fused sequence in the deleted haplotypes (fused SDs). We conducted this analysis for both humans and chimpanzees by means of sliding windows of 500 bp  together with the segmental duplication on the 5′ of the GSTM1 (SD1) and segmental duplication on the 3′ of the GSTM1 (SD2). Taking the gene conversion detected in this locus . This is true, even when we do not consider that two (not only one) of the breakpoints of GSTM1 deletion coincide in humans and chimpanzees. Moreover, the likelihood of this breakpoint sharing is even less given that the GSTM1 deletion is polymorphic in both chimpanzees and humans.Next, we aimed to evaluate whether the deletion sharing between humans and chimpanzees that we observed for the GSTM1 deletion. We found that only 12 of the 1713 polymorphic chimpanzee deletions overlap with 42,441 human deletions  that have maintained these polymorphic deletions remain a highly important next venue of research.Therefore, we wanted to know whether other polymorphic deletions with similar breakpoints are shared among humans and chimpanzees. To do this, we compared 1000 Genomes deletion polymorphism data  with polGSTM1 locus, including multiple gene conversion and structural variation events. By doing so, our work sheds light on the evolutionary diversification of a metabolizing GSTM gene family.Here, we scrutinized the evolution of the It is important to make a side note with regards to alignments because multiple analyses we used depend on the accuracy of these alignments. As mentioned earlier, the sequences of both segmental duplications and the fused-segmental duplications observed in the deleted chromosomes are very similar to each other  mutation rate. Even then, this rate is more than 100 times higher than the average nucleotide substitution rate [GSTM locus in primates.If these two deletions have happened in humans and chimpanzees independently after the species divergence, the lower bound on the mutation rate for the deletion can be calculated as 2/12.6 myr = 1.58 × 10ion rate  and at lion rate . OverallGSTM1 locus that we describe in this study harbors one of the few common gene deletions found in the human genome [GSTM1 and the other overlapping another metabolizing gene, the UGTB17. Indeed, structural variants observed in metabolizing gene families, including the UGT [CYP [NAT [The n genome . Our fin the UGT , 50, theUGT [CYP  and the CYP [NAT , contribCombining these insights, we argue that our findings can be generalizable to gene families that are comprised of similar, tandemly-located genes with environmental interaction functions. There is accumulating evidence that almost all such gene families have been rapidly evolving through complex structural variations , creatinPan troglodytes verus and a Pan Troglodytes troglodytes, and seven human samples ) were used in this study for the sequencing. The human samples were collected after informed consent was obtained. DNA samples used for sequencing are stored in the Primate Cell & DNA Repository of Unit of Human Biology & Genetics, Department of Biological Sciences, Graduate School of Science, The University of Tokyo. Four Chimpanzee DNA for ddPCR and Read-depth method were obtained from Coriell . Detailed information of the sample was described in [A total of 37 unrelated chimpanzees  and that of the entire genome and divided the depth in the GSTM1 by the depth of the entire genome.To genotype the eated by . We calchttp://www.ddbj.nig.ac.jp/). Sequencing analyses were conducted by Eurofin Genomics. The sequences generated during the current study are available in the DDBJ (http://www.ddbj.nig.ac.jp/) and the accession numbers are LC312398-LC312411).Fused segmental duplications of seven heterozygous chimpanzees and humans  were sequenced by primer walking method . Of the 2770 chimpanzee polymorphic deletions, we were able to lift-over 1713 deletions. We compared the 1713 deletion from chimpanzee data and human deletions reported in phase3 1000 genomes  [To evaluate the rarity of deletion polymorphisms shared between humans and chimpanzees, we took deletion polymorphism data of chimpanzees from a previous paper , then liGSTM gene sequences in primates, we modified a pipeline described earlier by [GSTM1 coding gene sequence was used as a reference and as input for tblastn [http://www.genome.jp/). The genomes that are used are as follows: Homo sapiens [Pan troglodytes [Pan paniscus [Gorilla gorilla gorilla [Pongo abelii [Nomascus leucogenys [Macaca mulatta [Callithrix jacchus [Tupaia belangeri chinensis [H. sapiens. This allows us to verify that these input primate sequences are indeed members of the GSTM family. In addition, the orthologs of different GSTM genes in nonhuman primates were identified based on the blastx results. If the top blastx hit was not a human GSTM sequence, the sequence was excluded. In the subsequent analysis, we considered only sequences that contain both GST N-domain and GST C-domain. We also annotate pseudogenes by documenting sequences with premature stop codons and gene truncations relative to the functional GSTM gene  using NCBI database (http://www.ncbi.nlm.nih.gov/gene).To obtain the rlier by . Specifi tblastn  searches sapiens , Pan troglodytes , Pan panpaniscus , Gorilla gorilla , Pongo ao abelii , Nomascuucogenys  Macaca m mulatta , Callith jacchus  and Tupahinensis ). The tbA similar approach was used in the detection of the segmental duplications. Briefly, the 2 kb highly similar segmental duplications in humans which were reported in  were useH. sapiens fused segmental duplication was used as input for blast search against primate genomes. Segmental duplication sequences in the reference genomes of H. sapiens, Pan troglodytes and Pongo abelii were conserved enough to align to each other. Nucleotide sequences of 7 human fused segmental duplications, seven chimpanzee fused segmental duplications that we sequenced along with the human, chimpanzee, orangutan segmental duplications retrieved from the reference genomes were aligned manually in MEGA7 [The entire sequence of a in MEGA7 . Some inin MEGA7  in the Min MEGA7  could noin MEGA7  and reprin MEGA7 .Based on the results of sliding window analysis, we divided the whole 12,541b segmental duplications into three parts . We conducted phylogenetic analyses of the sequences of the segmental duplications using ML methods in MEGA7 with the following models: 100 bootstrap replications, general time reversible model of nucleotide substitution, gamma distribution of rates, partial deletion with cutoff 95%, Nearest-Neighbor-Interchange, Make initial tree automatically .GSTM1 deletion is similar in chimpanzees and humans with about 4000 base pairs resolution ng MAFFT . Figure Additional file 2:Sequence alignment of the GSTM1 region of 7 deleted humans and 7 deleted chimpanzees and reference genomes of both species. (TXT 252 kb)


Although international medical graduates (IMGs) are essential in health care service delivery, a gap exists in the literature about how IMGs are selected into psychiatry residency programs in Canada. The purpose of this study was to identify the relative weight or importance that Canadian program directors (PDs) of psychiatry place on certain selection criteria when matching IMGs into residency programs.We electronically distributed a web-based questionnaire to 16 university residency program directors of psychiatry in Canada. Program Directors were asked to rate the importance of 43 selection criteria using 5-point Likert Scales. Criteria were grouped into six domains: academic criteria, extracurricular activities, supporting information, behavioural issues of concern, medical school country, and other education. Mean total values for each set of criteria were calculated and used to create rank orders within each domain.Eight out of 16 program directors responded. Our analysis indicated that academics and behavioral issues of concern were the most important selection criteria.Our findings provide valuable insight about the perspectives of Program Directors toward IMGs who apply for psychiatry residency programs in Canada. Further studies are needed to better understand which criteria contribute to IMGs’ performances as psychiatric residents. International medical graduates (IMGs) are individuals who received their medical training outside of Canada, including immigrant physicians and Canadians who studied abroad.2Historically, the majority of IMGs in Canada emigrated from Britain, Ireland, and South Africa, countries where training and accreditation systems are comparable to Canada’s.3Although IMGs are integral to the Canadian physician workforce, research suggests that when compared to their Canadian-trained counterparts, IMGs do not perform as well during their residencies and Royal College specialty examinations.1The process of selecting candidates for residency is often subject to unpredictable factors such as program funding changes and competition within a specialty.7Despite concerns regarding differences in competency between IMGs and CMGs during postgraduate training,This was a descriptive study that involved the administration of a modified version of the Residency Selection Criteria Questionnaire (RSCQ) to psychiatry PDs in Canada. The RSCQ was originally developed by Wagoner and SurianoFor the academic criteria domain, PDs were asked to rate the level of importance  for 15 items that related to academic performance, including clerkship narratives, grades for courses required during clerkships and for electives, academic awards, research experience, and examination results. As both Wagnoner and SurianoThe importance of Medical Council of Canada Qualifying Examinations (MCCQE) scores was evaluated using two items – one focused on the scores for Part I of the examination and the other on the score for the complete examination (Parts I and II). Additionally, PDs were specifically asked to evaluate the importance of having done observerships and research with Canadian physicians.PDs were also asked to evaluate the importance  of experience in three broad categories of extra- and co-curricular activities: leadership roles, community service, and experience in global health.For other non-academic criteria, PDs also rated the value of having various supporting information in the application (1=Disagree to 5= Agree), including personal statements and curriculum vitaes (CVs), and letters of recommendation and reference. Additionally, they weighted the importance of eight issues of concerns (1=No concern to 5=Very concerned) related to applicants’ personal background and academic records. Examples of these items are: disciplinary action during medical school, extended leave from medicine, and failed exams and courses. PDs were also asked to describe the relative importance  of where IMGs did their medical training and whether they had other graduate degrees.The questionnaire also contained a section to collect general information about the PDs’ residency programs. In addition, PDs were asked to provide a qualitative response on what they perceived to be the most important factor in selecting IMGS for interviews which, historically, have been a key component for resident selection in psychiatry.15After institutional ethical approval was obtained, an invitation to complete the RSCQ was delivered via email to 16 Canadian post-graduate PDs of Departments of Psychiatry, from September 1, 2013 to December 31, 2013. The email contained a link to a secure website where directors were requested to complete the RSCQ questionnaire. The questionnaire was completed anonymously and no personal information was recorded or requested of the individuals completing the questionnaire. To maximize the total number of completed surveys, an email reminder was delivered every 30 days, on three separate occasions, after the initial invitation.Data from completed questionnaires were downloaded and basic descriptive analysis  was used to examine the data. Mean values for each questionnaire item were calculated and used to create rank orders within each domain. All analyses were performed using SPSS V. 17.0 statistical software.Fifty percent (8 of 16) of surveyed PDs responded to the questionnaire. The mean total for issues of behavioral concern was 32.4 (± 4.0). The top four criteria that were scored of highest importance were: disciplinary action in medical school (4.9 ± 0.4), absence from medical school for more than three years (4.7 ± 0.5), receiving a failure in a required clinical clerkship (4.4 ± 0.8), and taking extended time to graduate for academic reasons (4.4 ± 0.7).Mean total score for where IMG applicants attended medical school was 11.8 (± 4.7). Whether their education was from the United Kingdom or Ireland (2.6 ± 1.1), Australia (2.6 ± 1.1), the Caribbean (2.6 ± 1.3), or from another country (2.1 ± 1.0) was of moderately low importance for PDs. The mean total for additional education was 5.4 (± 1.6) with having a PhD (2.1 ± 0.8) ranking highest, followed by MBA (1.6 ± 0.5), or MPH (1.6 ± 0.5).Seven of the eight responding PDs responded to the question that asked about the most important consideration in selecting IMGs for interviews. Their answers were:Genuine interest in psychiatry and graduation from medical school within the past five yearsGood fit with the programMedical Council of Canada Evaluating Examination plus composite evaluation of filePlace of training and time since graduationGenuine interest in psychiatryRecent, successful clerkship-level work in a Canadian teaching hospitalOverall applicationThe present study examined the relative importance that PDs from Canadian psychiatry residency programs have assigned to various selection criteria when matching IMGs into their programs. Although this was a preliminary study, responses from the questionnaires were generally consistent with previously published reports on the most important selection criteria for other residency programs.The most important selection criterion was issues of behavioral concern and among them, disciplinary action in medical school ranked the highest. Papadakis, et. al.Other important criteria within issues of behavioural concern were receiving a failure in a required clinical clerkship and failing two exams prior to passing. These criteria assessed candidates’ clinical skills and performance and have been shown to be highly valued.7The weights assigned to issues of behavioural concern demonstrate how greatly the PDs value academic criteria during the selection process. The issues that were assessed as most important focused on students’ academic performances either directly, such as failure in a required clinical clerkship, or indirectly, such as extended absence from medicine or extended time to graduate for academic reasons. When examined collectively, the emphasis on academic selection criteria over non-academic selection criteria may reflect PDs desire for accurate and objective assessments of the academic and clinical potential of candidates.10Research and publication experience was ranked very low, which again aligned with the literature.10Responses for non-academic domains suggest that PDs desire to obtain a well-rounded understanding of IMG applicants as individuals. Having this knowledge will likely help PDs better predict candidates’ future success as psychiatry residents. Letters of support from faculty members, for example, provide valuable insight as to how applicants are perceived.There are several limitations to our study. Preliminary analysis of the findings suggests there is little difference in the selection criteria between IMGs and CMGs; however, our study is limited by a small sample size due to a 50% response rate from Canadian psychiatry program PDs. As voluntary response bias is an inevitable limitation of surveys,Our study provides valuable insight into the selection process of IMGs into psychiatry residency programs in Canada, specifically at describing the relative levels of importance that PDs have placed on various criteria. Although this study was descriptive in nature, our findings contribute to a significant gap in the literature by providing important information about the perspectives of PDs in psychiatry. This is much needed given the increasing diversity of applicants for postgraduate training, especially from those who have trained outside of North America. The selection process for residency has thus become more complex, especially in psychiatry, which, out of all medical specialties, has the second highest number of IMGs.15Results of this study may help IMGs learn more about the selection process for Canadian residency programs, assist in strengthening their applications, and help them make better informed decisions about their careers. This information may also be useful for program directors, deans and educators in counseling IMG applicants and in developing their institutional curricula for IMGs.Lastly, this study highlights the need for future research, specifically which selection criteria best predict residents’ performance. Future studies should obtain a higher response rate, potentially by using a variety of methods to contact PDs. For example, in Green et al.’s study,


A high-density SNP panel  was used for genotyping. After genomic data quality control, 437,197 SNPs from 761 animals were available, of which 721 had phenotypes for LMA, 669 for BF, and 718 for RF. The SNP solutions were estimated using a single-step genomic BLUP approach (ssGWAS), which calculated the variance for windows of 50 consecutive SNPs and the regions that accounted for more than 0.5% of the additive genetic variance were used to search for candidate genes. The results indicated that 12, 18, and 15 different windows were associated to LMA, BF, and RF, respectively. Confirming the polygenic nature of the studied traits, 43, 65, and 53 genes were found in those associated windows, respectively for LMA, BF, and RF. Among the candidate genes, some of them, which already had their functions associated with the expression of energy metabolism, were found associated with fat deposition in this study. In addition, ALKBH3 and HSD17B12 genes, which are related in fibroblast death and metabolism of steroids, were found associated with LMA. The results presented here should help to better understand the genetic and physiologic mechanism regulating the muscle tissue deposition and subcutaneous fat cover expression of Zebu animals. The identification of candidate genes should contribute for Zebu breeding programs in order to consider carcass traits as selection criteria in their genetic evaluation.The purpose of this study was to identify genomic regions associated with carcass traits in an experimental Nelore cattle population. The studied data set contained 2,306 ultrasound records for  Bos indicus (Zebu) breeds and their crosses with Bos taurus. Even though Zebu animals have adaptive advantages to tropical conditions over Taurine breeds, these animals still have lower reproductive efficiency, lower beef tenderness, higher age at slaughtering, lower proportion of fat in the carcass [Beef cattle production in tropical and subtropical regions is predominantly based on  carcass ,2. The clongissimus muscle area (LMA) is an efficient indicator of the carcass yield, carcass muscularity [longissimus muscle acts minimizing evaporative weight loss of the carcass in the cooler besides been an efficient carcass-finishing indicative [Bos taurus breeds [The cularity , carcasscularity . The subdicative . Furtherdicative . In addis breeds , 8.Favourable genetic correlations between subcutaneous fat and reproductive traits reported by Caetano et al.  indicateThe use of genomic information has been a standard procedure for genetic evaluation in animal and plant breeding programs in order to improve quantitative traits. This procedure has been especially helpful for traits that are hard or expensive to measure and consequently not routinely recorded. Genome-wide association studies (GWAS) aim to identify regions in the genome that are associated with phenotypes and have been applied in many traits including carcass traits . A commolongissimus muscle area (LMA), subcutaneous rump fat thickness (RF), and subcutaneous backfat thickness (BF) in an experimental Nelore cattle population.Most of genomic studies for carcass traits have been applied on Taurine breeds in temperate regions . MoreoveThe analysed Nelore cattle data set was provided by the APTA Beef Cattle Center—Institute of Animal Science (IZ), Sertãozinho, SP, Brazil. This herd has three experimental lines: a selection line (NeS) which has been selected for yearling weight since 1978; the traditional line (NeT) which has been submitted to the same selection criterion as NeS but, eventually, receives animals from other herds; and a control line (NeC) selected for average yearling weight (stabilizing selection). Ethics committee of the School of Agricultural and Veterinarian Sciences (FCAV), Sao Paulo State University (UNESP) and APTA Beef Cattle Center—Institute of Animal Science approved this study.longissimus muscle area (LMA), 1,832 for backfat thickness (BF), and 1,830 rump fat thickness (RF). The ultrasound animals were born from 1996 to 2013. The data set contained pedigree information on 9,529 animals, of which 2,306 had ultrasound records for The herd management consisted on keeping the animals on pasture until seven months of age, when they were weaned. After this period, males and females  were submitted to a performance test on feedlot.Gluteus Medius and Biceps Femoris muscles, which is located between ilium and ischium bones.Serial ultrasound data were collected in vivo at 12 and 18 months of age for males and females, respectively. The difference among the age of phenotypic ultrasound measurement was mainly due the differed after weaning management applied for each sex. The animals were scanned by the PIE MEDICAL—Aquila equipment with probe of 7 inches and 3,5MHz, and then, the images were analysed using the Echo Image Viewer 1.0  software. The probe was perpendicularly positioned between the 12th and 13th ribs on the left side to obtain the LMA and BF phenotypes. To obtain the RF phenotype, the probe was positioned at intersection of 2EDTA Vacutainer blood collection tubes (BD Diagnostics) by jugular venepuncture. These samples were inverted to mix and prevent clotting, immediately placed in isothermic boxes, and transferred to the laboratory. During sampling animals were handled by qualified professionals who observed the procedure of well-being and safety of all animals.DNA was extracted from blood samples which were collected in 9 ml KA high-density SNP panel  was used for genotyping. SNP markers with minor allele frequency (MAF) and call rate less than 2% and 98%, respectively, were excluded. Also, samples with a call rate less than 90% were not considered in analyses. After genomic data quality control, 437,197 SNPs from 761 animals were available, of which 721 had phenotypes for LMA, 669 for BF, and 718 for RF trait .2a and σ2e are total additive genetic and residual variances, respectively, a is the vector of direct additive genetic effects, e is a vector of residual effects, and H is a matrix that combines pedigree and genomic relationships, and its inverse consists on the integration of additive and genomic relationship matrices, A and G, respectively [A-1 is the inverse of relationship matrix based on pedigree information, G-1 is the inverse of genomic relationship matrix, which was constructed and scaled as described by VanRaden [A-122 is the inverse of pedigree-based relationship matrix for genotyped animals. The H matrix was built scaling G based on A22 considering that the average of diagonal of G is equal to average of the diagonal of A22 and average of off diagonal G is equal to average off diagonal A22.The (co)variance components and genetic parameters were estimated by Bayesian inference, considering a linear single-trait animal model. Direct additive genetic and residual effects were included as random effects. Analyses were performed using GIBBS2f90 , 15. TheVanRaden , and A-1Y is the vector of phenotypic observations, X is an incidence matrix of phenotypes and fixed effects, b is the vector of fixed effects, that included contemporary groups (sex and year of birth), and age of ultrasound measurement as a covariable (linear and quadratic effects), Z is an incidence matrix that relates animals to phenotypes. The principal components analyses made based on genomic relationship matrix (G) revealed population sub-structure  and ungenotyped (an) animals as describe by Wang et al. [Z was a matrix that related genotypes of each locus and u was a vector of marker effects. The variance of animal effects was assumed as:D was a diagonal matrix of weights for variances of markers (D = I for GBLUP), G*) was built with no weight.The analyses were performed using the single-step GWAS methodology  considerg et al. , with thλ was defined by VanRaden et al. [M was the number of SNPs and pi was the frequency of the second allele in the i-th SNP.Thus, the SNP effects were obtained following equation, as described by Wang et al. :ȗ=λDZ’Gn et al.  as a norD to estimate the SNP effects: 1. D = I; 2. To calculate GEBVs for all animals in data set using ssGBLUP; 3. To calculate the SNP effect: ȗ = λDZ’G*-1ȃg; 4. To calculate the variance of each SNP: di = ûi22pi (1—pi), where i is the i-th marker; 5. To normalize the values of SNPs to keep constant the additive genetic variance; 6. To calculate the G matrix; 7. Exit, or loop to step 2.The following iterative process described by Wang et al.  was usedai is genetic value of the i-th region that consists of 50 consecutive SNPs, σ2a is the total genetic variance, Zj is vector of SNP content of the j-th SNP for all individuals, and ûj is marker effect of the j-th within the i-th region.The effects of markers were obtained by 2 iterations from step 2 to 6 as showed by Wang et al. . The perAnalyses were performed using BLUPf90 family software  modifiedhttp://www.ncbi.nlm.nih.gov) in UMD3.1 bovine genome assembly and Ensembl Genome Browser (http://www.ensembl.org/index.html). The classification of genes according to its biological function, identification of metabolic pathways and enrichment of genes was performed using the "Database for Annotation, Visualization and Integrated Discovery (DAVID) v. 6.7” (http://david.abcc.ncifcrf.gov/), GeneCards (http://www.genecards.org/), and UniProt (http://uniprot.org).The chromosome segments that explained more than 0.5% of additive genetic variance were selected to explore and determine possible quantitative trait loci (QTL). The Map Viewer tool of bovine genome was used for identification of genes, available at "National Center for Biotechnology Information" . The first cluster from enrichment analysis for LMA was related to processes that stop, prevent or reduce the rate of cell death by apoptotic process . The clusters obtained by the genes enrichment analysis for BF were related to ATP-binding, transcription regulation, and protein amino acid phosphorylation (S2 Table). Many genes that are related to lipids and fat expression were found in the windows associated with BF.A total of 42 known genes and 23 uncharacterized genes  were found in those windows associated to BF . Some of those regions have been reported to be associated with lipid production (Table 5). These findings confirm the polygenic nature of fat deposition. The clusters obtained by the genes enrichment analysis of RF are related to ATP binding, response to hormone stimulus and intracellular signalling cascade (S3 Table).A total of 32 known genes and 19 uncharacterized genes  were found in the windows associated to RF  was associated to both fat deposition traits, BF and RF, where the H matrix to the structure of analysed population. Heritability values showed that a great part of phenotypic variance was due to additive genetic effects. Thus, it is important to identify the genes involved in these traits expression, especially major effects genes.The confidence interval for posterior distribution of heritability was very accurate for all studied traits; the range was always within 4 points from the mean. This narrow posterior distribution indicates a good adjustment of the ALKBH3 and HSD17B12, which are related in fibroblast death and metabolism of steroids, were found associated with longissimus muscle area.Several known genes that have their functions previously reported associated to the expression of energy metabolism were confirmed associated with fat deposition in this study. In addition, genes such as the ALKBH3 gene (found in a window that most explained additive genetic variance of LMA located in BTA15) codifies an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes [HSD17B12 gene is located. This gene is a member of the hydroxysteroid dehydrogenase superfamily, involved in the metabolism of steroids, retinoids, bile and fatty acids that apparently can be related in metabolic pathways involved in tumor [The ed genes . Even thed genes  reportedin tumor . ConsideHTR2B gene, located on BTA2 (Table 4), was found in a window associated to BF. This gene has been previously associated to fat deposition in humans [ALDH3B1 and CHKA genes. The ALDH3B1 gene had its function related to oxidation of lipid-derived aldehydes generated in the human plasma membrane [CHKA gene is related to lipid biosynthesis and it has been reported differentially regulated in mice by high fatty acids levels [The n humans  and it imembrane  and is amembrane . The bios levels . Even thXKR4 gene (placed in a window associated with BF located on BTA14) was previously reported by Porto Neto et al. [XKR4 gene (P < 0.001), one of the SNPs alone explained 5.9% of the additive genetic variance. This gene has also been reported as a candidate gene for residual feed intake, average daily feed intake and average daily gain [XKR4 gene participates in the regulation of prolactin secretion in cattle.The o et al.  to be asily gain . In addiily gain  suggesteTable 4 and Table 5). In this associated window were found the PLAG1, CHCHD7, MOS, RPS20, LYN, and RDHE2  genes. There are evidences that these genes are directly associated with the expression of many traits in beef cattle. Independent studies have reported association between PLAG1 gene with fat, residual feed intake, carcass, performance and reproductive traits in beef cattle breeds including Nelore, Japanese Black and Korean cattle [PENK gene, also located in a window on BTA14 (at 25.20–25.49 Mb), was previously reported associated with residual feed intake in a swine population [PENK and PLAG1, are located (at 24.87–25. 10 Mb and 25.20–25.49 Mb of BTA15) explained a great part of additive genetic variance of both fat deposition traits, BF and RF. Thus, once they are juxtaposed and with possible physical disequilibrium, it is hard to say whether these genes have independent function or the associated windows where they are located are in linkage disequilibrium. Karim et al. [PLAG1-CHCHD7 intergenic region affected the bovine stature in a European breed. Nishimura et al. [A window located on BTA14 (at 24.87–25.11 Mb) was associated to both fat deposition traits, BF and RF  and genetic (0.79 ± 0.05) correlated  differinCAPN5 and MYO7A (located at 57.29–57.43 Mb of BTA15) genes are associated with blood cholesterol level in humans and with abdominal fat in chicken [LUM gene (located at 20.99–21.12 Mb of BTA5) was related with omental fat deposition in humans [BAI3 gene, placed in a region of BTA9 acts on the fusion of myoblast and it is expressed on extracellular matrix [Genes related with fat deposition were found in windows that explained a great part additive genetic variance of RF trait. The  chicken , 43. In n humans . The BAIr matrix  that parRPS6KB1 gene (located at 10.84–11.30 Mb of BTA19) is sub expressed in skeletal muscle of calves which were experimental fed with high levels of protein. In addition, the DCN gene (located at 20.99–21.12 Mb of BTA5) operates in intramyocellular space co-acting with myostatin gene [Some others genes had their expression identified in skeletal muscle and performance capability on adipose tissue, since that tissue are juxtaposed and have many genes with common acting. According to Wang et al. , the RPStin gene  and it itin gene .longissimus muscle area, backfat thickness and rump fat thickness traits are governed by many variants with small effects, the results in the present study confirm the polygenic effect of these traits. On the other hand, it is important to highlight that while several DNA regions with larger effects have been detected, some of these may not be due to variants but could be mark chromosome segments from important ancestors or just be sampling noise. In this study, 437,197 SNPs effects were predicted from about 700 data points (DGVs). With so few points, there is a statistical possibility that a limited number of SNP could provide good prediction as shown by cross-validation just by chance. Stam [Considering that ce. Stam  pointed ALKBH3 and HSD17B12 are more likely to be related to longissimus muscle area, as well as the PLAG1, CAPN5, MYO7A and XKR4 genes, which are possibly associated with fat deposition in Nelore cattle. Thus, the identification of these genes should contribute for future studies to validate them to use as candidate genes for Zebu breeding programs.The results presented here should help to better understand the genetic and physiologic mechanism regulating the muscle tissue deposition and subcutaneous fat cover expression of Zebu animals. The analysed data set contained information from proved animals of an experimental herd, which have progenies participating in many breeding programs in several regions of Brazil. Despite the polygenic nature of the studied traits, some genes found in the associated windows, such as S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The current study investigated the interactive effects of individual differences in automatic emotion regulation (AER) and primed emotion regulation strategy on skin conductance level (SCL) and heart rate during provoked anger. The study was a 2 × 2 [AER tendency (expression vs. control) × priming (expression vs. control)] between subject design. Participants were assigned to two groups according to their performance on an emotion regulation-IAT (differentiating automatic emotion control tendency and automatic emotion expression tendency). Then participants of the two groups were randomly assigned to two emotion regulation priming conditions (emotion control priming or emotion expression priming). Anger was provoked by blaming participants for slow performance during a subsequent backward subtraction task. In anger provocation, SCL of individuals with automatic emotion control tendencies in the control priming condition was lower than of those with automatic emotion control tendencies in the expression priming condition. However, SCL of individuals with automatic emotion expression tendencies did no differ in the automatic emotion control priming or the automatic emotion expression priming condition. Heart rate during anger provocation was higher in individuals with automatic emotion expression tendencies than in individuals with automatic emotion control tendencies regardless of priming condition. This pattern indicates an interactive effect of individual differences in AER and emotion regulation priming on SCL, which is an index of emotional arousal. Heart rate was only sensitive to the individual differences in AER, and did not reflect this interaction. This finding has implications for clinical studies of the use of emotion regulation strategy training suggesting that different practices are optimal for individuals who differ in AER tendencies. Automatic emotion regulation (AER) is characterized by goal-driven changes to one's emotional experience without making a conscious decision to do so and without engaging in deliberate control , has positive effects on emotion regulation has been found in studies involving measures as diverse as resting prefrontal spontaneous alpha asymmetry and self-reported  will show the lowest autonomic activation (lowest SCL and heart rate) and participants with an automatic emotion expression tendency in the expression priming condition (AEET/EP) will show the highest autonomic activation. Participants with an automatic emotion control tendency in the expression priming condition (AECT/EP) and participants with an automatic emotion expression tendency in the control priming condition (AEET/CP) are expected to show intermediate levels of autonomic activation.The protocol of this study was approved by the Institutional Review Board of the Department of Psychology of Renmin University of China. Informed written consent was obtained from each subject before the experiment.M = 21, SD = 4.4, 46 males, and 48 females) from Renmin University of China volunteered participation in the study and were compensated with 30 Chinese Yuan. Forty-eight participants were selected into the automatic emotion control group and 48 participants were assigned to the automatic emotion expression group . Of the 48 participants with automatic emotion control tendencies, half were assigned randomly to receive emotion control priming and half were assigned to receive emotion expression priming. Of the 48 participants with automatic emotion expression tendencies, 24 were randomly assigned to emotion control priming, and 24 to emotion expression priming, however, two participants in the latter group did not complete the experiment leaving 22 in this condition. The 24 participants with automatic emotion control tendency in the emotion control priming condition, included 13 males and 11 females ; the 24 participants with automatic emotion expression tendency in the emotion control priming condition, included 12 males and 12 females ; the 24 participants with automatic emotion control tendency in the emotion expression priming condition, included 12 males and 12 females ; the 22 participants with automatic emotion expression tendency in the emotion expression priming condition, included 11 males and 11 females . Thus, the experimental design involved four groups, AEET/EP, automatic emotion expression tendency/expression priming; AEET/CP, automatic emotion expression tendency/control priming; AECT/EP, automatic emotion control tendency/expression priming; AECT/CP, automatic emotion control tendency/control priming.Ninety-four students  × priming (expression vs. control)] between subject factorial design.Participants arrived at the laboratory individually and completed the Chinese version of the Emotion Regulation- Implicit Association Test (ER-IAT), which was adapted from Mauss et al. . ParticiA sentence unscrambling task was used to prime emotion regulation goals (control or expression). Each participant received 10 sets of five words and was asked to form a sentence using four of the five words provided per set. In the emotion expression priming condition, nine sets contained a word related to emotion expression whereas in the emotion control priming condition nine sets contained a word related to emotion control. All participants also received one filler set.The 18 emotion control or expression related words used in the priming task were selected from 40 emotion regulation related words rated by 30 undergraduate students who majored in psychology. Nine emotion control-related words (See Appendix A) were selected that received a rating of 3 or more on a 5-point scale for the dimension of emotion control. Another 9 expression-related words (See Appendix A), which received a rating of 3 or more on a 5 points scale for the dimension of emotion expression, were selected as the emotion expression-related words.After the sentence unscrambling task, participants were instructed to complete a backward subtraction task, which required participants to complete three successive subtractions for 1 min each, 7 from 18,652, 7 from 87,611, and 7 from 20,134. The experimenter interrupted participants and blamed them for slow performance while participants were performing the subtraction. The experimenter said “Be quick! You are too slow!!!” This anger provocation task lasted 3 min for each participant. Participants completed the Chinese version of the PANAS  using Acknowledge 3.9.1 data acquisition and processing software on an IBM-compatible Pentium computer. Skin conduction level and an electrocardiogram (ECG) were recorded continuously from the beginning of the baseline film to the end of the backward subtraction. The ECG was collected using three disposable electrodes, placed on the participant's right forearm, and 10 cm above the right and left ankle. Electrodes were placed after the skin was wiped with alcohol and scrubs. The ECG was recorded with an ECG100C amplifier with a band pass from 0.5 to 35 Hz, and a gain of 500. The ECG was converted off-line to heart rate (HR) in beats per minute by Biopac processing software (Acqknowledge 3.9.1). Skin conductance was recorded with a GSR100C module, amplified to 5 μSiemens/V, with a DC high pass and a 1 Hz low pass filter. Two electrodes filled with electrode gel (GEL100) were applied to the tips of the index and middle fingers after cleaning the skin. To assess the effect of the anger provocation, the mean SCL and heart rate were derived from an interval that started at the beginning of the subtraction and ended after the first blame was delivered.Separate 2 × 2 factorial ANCOVAs were conducted on anger ratings derived from an adapted Chinese version of the PANAS  = 14.803, p < 0.01, ηp2 = 0.143, with higher anger reported in the expression priming condition than in the control priming condition. No other significant effects were found (ps < 0.05). The ANCOVA showed no significant effects for the rating data after priming (ps < 0.05). The means and SD of anger ratings were shown in Table As shown in Figure F, 94 = 14F = 6.192, p < 0.05, ηp2 = 0.065, with larger SCL in the expression priming condition than in the control priming condition, and AER tendency, F = 11.864, p < 0.01, ηp2 = 0.118, with lager SCL for individuals with automatic emotion expression tendency than those with automatic emotion control tendency. These main effects were qualified by a two way interaction, F = 4.428, p < 0.05, ηp2 = 0.047 . However, the SCL of individuals with automatic emotion expression tendencies showed no difference in the automatic emotion control priming or the automatic emotion expression priming condition (p < 0.05).An analysis of the SCL data after the anger provocation revealed main effects of priming condition, F = 7.983, p < 0.01, ηp2 = 0.082. The SCL in the expression priming condition was higher than in the control priming condition. No other effects were found (ps < 0.05). The means and SD were presented in Table An analysis of the SCL data after the priming revealed a main effect of priming condition, F, 94 = 7.F = 13.815, p < 0.001, ηp2 = 0.134. The participants with automatic expression tendencies had higher heart rates than participants with automatic control tendencies (p < 0.01). The same pattern was also found for the heart rate after priming, main effect of automatic emotion expression tendency, F = 8.171, p < 0.01, ηp2 = 0.084. Participants with automatic expression tendencies had higher heart rates than participants with automatic control tendencies after priming (p < 0.05). The means and SD of heart rate were presented in Table Heart rate data are displayed in Figure F, 94 = 13This study investigated whether individual difference in AER and an AER priming manipulation interact in their effects on emotion regulation during an anger provocation. We found that individuals with automatic emotion control tendencies primed to control emotion showed a lower SCL than individuals with automatic emotion control tendencies primed to express emotion only after anger provocation, not in the priming period. However, the SCL of individuals with automatic emotion expression tendencies showed no difference in the control priming or the expression priming condition. This pattern supports the hypothesis that individual differences in AER interact with the primed emotional regulation strategy. Heart rate of individuals with automatic emotion expression tendencies was higher than that of individuals with automatic emotion control tendencies in response to the anger provocation regardless of the emotion regulation priming condition. These results indicated that the SCL was sensitive to the interactive effects of individual differences in AER and AER priming. These results also provided an indication that heart rate might be responsible for individual differences in AER but not AER priming.Emotion regulation priming differentially affected SCL for persons with automatic emotion control tendencies, but had a uniform effect for persons who displayed automatic emotional expression tendencies. This result revealed that the SCL of the group of automatic emotional control individuals would be changed by priming regulation goals. In the anger provocation, they increased SCL under the emotion expression priming and decreased SCL under the implicit goal of emotion control. The SCL of the group of automatic emotion expression individuals were very high either in emotion control priming or in emotion expressing priming. SCL, which reflects sympathetic nervous activity, is a reliable and sensitive index of emotional arousal and arousal changes , the Fundamental Research Funds for the Central Universities and the Research Funds of Renmin University of China (15XNL029) granted to JZ, and partly supported by the Australian Research Council (DP150101540) to OL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While we are currently unable to predict which patients with MGUS will develop MM, the Mayo group has devised a risk stratification system to help in predicting patients with a higher risk of disease progression4. Smoldering multiple myeloma (SMM) is distinguished from MGUS by a higher burden of plasma cells and an increased risk of progression to MM5. There is currently no treatment for MGUS or SMM, and no treatment has been proven to reduce the risk of disease progression, though SMM remains an area of active research.Multiple myeloma (MM) lies along a spectrum of plasma cell disorders and represents the incurable malignant phase of disease. MM is felt to be uniformly preceded by an asymptomatic clonal disease called monoclonal gammopathy of undetermined significance (MGUS) that carries a lifelong risk of disease progression6. While patients with MGUS and SMM are generally felt to be asymptomatic, these patients do have an increased risk of bone fracture, linked to decreased bone strength and altered bone architecture9. Vitamin D is a key regulator of bone metabolism associated with progressive disease in several malignancies, including MM12. Dysregulation of RANKL and OPG correlates with disease activity and decreased survival, and has also been linked to altered bone metabolism in MGUS13. Based on the abnormal bone metabolism observed in patients with MGUS and the central role for vitamin D in bone metabolism, we hypothesized that abnormal bone metabolism would correlate with disease risk in MGUS and that vitamin D supplementation would improve bone metabolism and markers of disease. We investigated this hypothesis by correlating the vitamin D level with the risk category of MGUS and markers of bone metabolism, RANKL and OPG. We further evaluated the impact of vitamin D supplementation on RANKL and OPG levels, as well as markers of disease activity, including serum protein electrophoresis (SPEP) and free light chains (FLCs).Alterations in bone metabolism, mediated in part through upregulation of receptor activator of nuclear factor κβ (RANKL) and the decoy receptor osteoprotegrin (OPG), via effects on osteoblasts and osteoclastogenesis are common to both MM and MGUS and may play a role in disease progression3 who were older than 18 years of age were recruited to this study from a plasma cell dyscrasia clinic between June 2012 and November 2014. Patients with a history of osteoporosis, or other bone diseases, bisphosphonate usage within 1 year or glucocorticoid use within 3 months, pregnant patients, patients on bile acid sequestrants, anticonvulsive, or antiretroviral therapy, and patients with a history of malignancy other than in situ cancers within 5 years were excluded. Patients were stratified into two groups based on risk of disease progression, those with 0 or 1 risk factor for progression (low risk or intermediate-1 risk) vs. those with 2 or 3 risk factors (intermediate-2 or high risk), or SMM. Per the Mayo risk stratification system, possible risk factors include a non-IgG isotype, an IgG paraprotein level >1.5 g/dL, and an abnormal FLC ratio4. Ethical approval for this study was granted by the University of Kansas Ethics Committee and all patients provided informed consent.In this open-label trial, 50 patients with MGUS or SMM per the International Myeloma Working Group criteriaOnly patients with 25 hydroxyvitamin D (25(OH)D) < 20 ng/mL were asked to take cholecalciferol 6000 IU daily for 8 weeks followed by 2000 IU daily. Patients purchased their own cholecalciferol from a local pharmacy and were asked to complete a medication log documenting daily compliance. Patients were followed for 12 weeks.Blood samples were collected as fasting morning labs at baseline and at 12 weeks after the initial blood sample. Full blood count, creatinine, serum paraprotein, serum FLCs, electrophoresis with immunofixation, and 25(OH)D levels were determined at each visit. Additional blood was collected in EDTA and stored at −80 degrees for plasma analysis of sRANKL, OPG, and interleukin-6 (IL-6). Total sRANKL (BioVendor), OPG (Raybiotech), and IL-6 (Raybiotech) levels were determined using enzyme-linked immunosorbent assay kits in duplicate with the lower limits of detection at 25, 1, and 0.7 pg/mL, respectively. All assays were calibrated using standard curves following the manufacturer’s instructions.t-test with a type I error rate of 0.05 . Group values are expressed as the mean±SEM. Group comparisons were made using the unpaired Student’s t-test. Data within groups, pre vs. post-therapy were compared using paired Student’s t-test. Statistical significance was assigned as a P value < 0.05.Patients were grouped according to the risk group of MGUS or SMM, and further analyzed as vitamin D-deficient vs. replete. The study planned to enroll 50 patients, 30 with low risk and 20 with low-risk MGUS to provide ~80% power to detect a 0.8 s.d. of the RANKL/OPG ratio between low- and high-risk patients for a two-sized, two-sample In all, 50 patients were enrolled into the study  with an increase in the iFLC  and RANKL . Figure A total of 32 71%) patients were either found to be vitamin D-deficient or had a history of vitamin D deficiency requiring vitamin D supplementation at baseline. Patients with vitamin D deficiency had higher levels of RANKL and the RANKL/OPG ratio than replete patients  had 25(OH)D levels < 20 ng/mL. Additionally, several patients within our study who were assigned to take cholecalciferol, failed to have restoration of their 25(OH)D levels. Despite medication logs, it is impossible to exclude poor compliance, seasonal variation, or malabsorption as a mechanism for the failure of cholecalciferol supplementation. However, the correlation between vitamin D status and bone metabolism independent of supplementation, suggests a possible difference in disease biology amongst patients whose 25(OH)D levels improve with supplementation vs. those who do not.We further demonstrate that RANKL and RANKL/OPG are increased with high-risk MGUS and SMM vs. low risk. These data support a link between disease activity and bone metabolism for patients without clinically relevant plasma cell disorders and may provide a mechanism for the bone abnormalities and increased risk of fracture that is seen in patients with MGUS. In our study, vitamin D deficiency was independently linked to bone metabolism. Our study was confounded by the high baseline rate of vitamin D supplementation within our patient population. Based on patient history and the observed 25(OH) D levels within our study, 71% of patients were vitamin D-deficient, consistent with prior data4. Within our study, supplementation with cholecalciferol improved 25(OH)D levels and correlated with a decrease in markers of bone metabolism and disease activity as measured by iFLC, FLC ratio, and the SPEP. Since baseline supplementation status was not correlated with disease activity, our study suggests a direct action of cholecalciferol on disease biology that may be mediated through improvements in bone metabolism.The iFLC, FLC ratio, and SPEP represent surrogates for disease activity in plasma cell dyscrasias, including MGUS and SMMIn our study, a large number of enrolled patients failed to complete the study. Likewise, it is impossible to determine if low 25(OH)D levels contribute to altered bone metabolism and disease progression or if alterations in disease biology lead to altered 25(OH)D levels. However, our data do suggest a role for vitamin D in plasma cell mediated bone disease and disease progression that should be further studied in a larger study across the spectrum of disease activity.Supplementary Table 1


Sphincter-preserving operations performed with bladder-preserving surgery and a cystourethral anastomosis (CUA) do not require a urinary stoma, but leakage from the CUA may develop. The aim of this study was to evaluate the efficacy of performing an additional flap operation.The subjects were 39 patients who underwent bladder-preserving surgery for advanced rectal cancer involving the prostate, between 2001 and 2015.32 of whom had a CUA and one of whom had a neobladder. Five of these 32 patients underwent an ileal flap operation, 2 underwent an omental flap operation, and 3 underwent an operation using both flaps.Leakage developed in 3 (30%) of the 10 patients who underwent additional flap operations, but in 14 (60.9%) of the 23 patients who did not undergo a flap operation. The mean periods of catheterization for the patients who suffered leakage were 31 weeks (8–108 weeks) in those without a flap and 16 weeks (8–20 weeks) in those with a flap. Four (33.3%) of the 12 patients with leakage after surgery without a flap had a period of urinary catheterization >30 weeks, and 2 (16.7%) had leakage of CTCAE grade 3. There were no cases of leakage after flap surgery.An additional flap operation may decrease the risk of leakage from a CUA. Abdominoperineal resection (APR) is the standard surgery for locally advanced rectal cancer located within 5 cm of the anal verge . With tuBetween January, 2001 and January, 2015, 39 patients with locally advanced primary and recurrent rectal cancer with clinical involvement of the prostate underwent extended colorectal resection, combined with radical prostatectomy, at the National Cancer Center Hospital East. All 39 patients were originally considered to be candidates for TPE or ISR with radical cystoprostatectomy. However, ultimately, the urinary bladder was preserved in 38 patients and a neobladder was reconstructed in one patient, to avoid TPE and radical cystoprostatectomy. Four patients agreed to undergo preoperative radiochemotherapy based on our previous protocol , althougTable All patients had a localized rectal tumor involving the prostate and seminal vesicle, or combined with carcinoma of the prostate, without evident distant metastases preoperatively. All resected specimens were examined clinicopathologically and involvement of adjacent organs and surgical margins, perioperative morbidity and mortality, locoregional control, overall survival (OS), and disease-free survival (DFS) were investigated. Informed consent was obtained from all patients and approval was given by the institutional review board. The study was performed in accordance with the Helsinki Declaration of 1975 and 1983.Bladder-sparing extended en bloc rectal resection combined with radical prostatectomy was considered if the tumor was adhered to the prostate or bladder, or if an adequate margin between the tumor and these organs seemed impossible to achieve, based on magnetic resonance imaging (MRI) and computed tomography (CT) findings. Patients undergoing this surgical procedure had a residual urinary bladder with available capacity of 50 ml and the possibility for CUA. Distant metastasis, wide-ranging involvement of the urinary bladder, prostate, and marked pelvic lymph node metastasis were generally considered contraindications for this operation, since preservation of the available urinary bladder was impossible in patients with wide-ranging involvement of the urinary bladder. Those patients underwent TPE.The surgical technique was as follows: after placing the patient in the lithotomy position, total mesorectal excision with lateral pelvic node dissection was performed . The ureters were located and carefully protected throughout the procedure, and the superior vesical artery was preserved bi- or unilaterally. The status of involvement of the rectum and base of the bladder was investigated at this point. After confirming the absence of wide-ranging involvement of the urinary bladder, bladder sparing surgery was deemed possible. The rectal cancer, prostate, and seminal vesicles were resected en bloc cooperatively, by colorectal and urological surgeons using the usual methods for radical prostatectomy and ISR or APR. APR was performed when safe surgical margins could not be obtained by ISR. Frozen section examination of the resected specimen was done to confirm cancer-free radial and distal margins, evaluate the extent of pelvic invasion, and determine whether limited resection was possible. If tumor invasion was suspected in the surgical margins on the intraoperative histological examination, the operative procedure was converted to TPE. After confirming preservation of the membranous urethra and bladder, the bladder neck was reconstructed and a CUA was created by urological surgeons Fig. . An ileaOn postoperative day 28, the strength of the CUA was assessed by cystography using 100–150 ml of standard contrast agent  diluted in 100–150 ml of 0.9% NaCl inserted under gravity into the bladder via the Foley indwelling catheter. Anteroposterior and oblique views were obtained after contrast instillation. The cystography findings were evaluated by an experienced urologist. Perianastomosis leakage was classified as no leakage or leakage. In a patient with no leakage, the catheter was removed the same day. For a patient with leakage, radiological follow-up was conducted after 1–3 months. The leakage grade was classified using the National Cancer Institute Common Terminology Criteria for Adverse Events  .n = 3; T4, N1: n = 1; T4,N0: n = 1) between 2001 and 2010 agreed to undergo preoperative radiochemotherapy using our old protocol, despite this treatment for resectable rectal cancer not being standard in Japan, even for patients undergoing TPE. These patients received 45 Gy to the whole pelvis over a 5-week period, followed by resection after 2 weeks. In addition, 5-fluorouracil (5-FU) was administered as a continuous infusion at 250 mg/m2/day during the radiotherapy to enhance the radiotherapeutic efficacy. Postoperative chemotherapy (5-FU/leucovorin therapy or FOLFOX) was offered to patients with pathological stage III disease.Five patients . Examinations were performed every 3 or 4 months for 2 years postoperatively, and every 6 months thereafter. These comprised clinical examinations; laboratory tests including tumor marker levels; lung, liver, and pelvic CT; and physiological assessment using anal manometry and uroflowmetry.The starting point for survival and recurrencefree intervals was the day of the operation, and data ofpatients who were alive or free of recurrence were censored at the last follow-up. OS was defined as the time from radical surgery until death from any cause. Local recurrence was defined as recurrence confined to the pelvis, and distant recurrence was defined as recurrence outside of the pelvis. Statistical analyses wereperformed using SPSS for Windows v. 11.0 J . OS and DFS curves were calculated using the Kaplan–Meier method.The present study was registered with the University Hospital Medical Information Network Clinical Trials Registry (no. 000010530). The institutional review broad approved the protocol for this study. An independent safety monitoring committee monitored the safety of the patients throughout the study period. The study was performed under the Declaration of Helsinki and Japanese Good Clinical Practice Guidelines.n = 18; U-LAR, n = 4) with radical prostatectomy, and 16 underwent APR with radical prostatectomy because the lower edge of the tumor was very close to the anal verge or the tumor involved the external anal sphincter. One patient underwent recurrent tumor resection and radical prostatectomy after APR.Twenty patients had localized tumors with clinical involvement of the prostate, three had clinical T3 lower rectal tumors with synchronous primary prostate cancer, and one underwent radical prostatectomy. Five patients had local recurrence with clinical involvement of the prostate. No procedures were converted to TPE because of inadequate margins. Twenty-two patients underwent anal SPOs , with histological cancerous invasion of the prostate in 11; a pT3 tumor with fibrosis or inflammatory changes surrounding the tumor in 15; and a pT2 tumor with the same histological changes in 4. The T1b patient, 2 of the 4 pT2 patients, and 1 of the 15 pT3 patients had synchronous primary prostate cancer, and it was difficult to separate the rectal tumors safely from the prostate during the operation in these patients. Curative resection was achieved in 38 patients by bladder-preserving surgery and in one patient by using a neobladder.Table A CUA leak was identified in 17 patients and required catheterization through the anastomosis site for 8 to 108 weeks postoperatively. Of the 10 patients who had additional flap operations, 3 (30.0%) suffered leakage, whereas of the 23 patients who had no flap operation, 14 (60.9%) suffered leakage (Table n = 1), multiple lung metastases (n = 4), multiple liver and bone metastases (n = 1), multiple lung and bone metastases (n = 1), and local re-recurrence (n = 2). The patient treated with a neobladder died of an abdominal injury sustained in a fall  of the 34 patients with primary disease, and the incidence of local recurrence was 26.7%  of 32 patients with a CUA. Similarly, Wiig et al.  found thThe method described in this study is a part of an operation for cancer; therefore, prognosis and recurrence are very important considerations. Similar procedures were described by Campbell et al.  in two pIn the present study, six patients (15.3%) suffered local recurrence during follow-up, including one recurrence in the presacral area. This local recurrence was unlikely to have been prevented had the patient undergone TPE. The patient underwent successful resection of the recurrent tumor, but died of multiple lung metastases 86 months after the initial operation. In another patient, local recurrence developed at the paravesical site. This patient also underwent resection of the recurrent tumor and remains re-recurrence free after 90 months. In a third patient, local recurrence developed at the parasacral site. Resection of the recurrent tumor was not possible and despite chemotherapy, the patient died 38 months after the first operation. In a fourth patient, local recurrence developed in the left internal obturator muscle. Again, tumor resection was not possible, but chemoradiotherapy was given and this lesion is in complete remission, with the patient being re-recurrence free after 60 months. The last two patients both had recurrent lesions in the pelvic area at their first operation in our hospital. One underwent resection of the recurrent tumor, but died 55 months after the operation; and for the other patient, resection of the recurrent tumor was not possible and he died 1 month after the operation.In 2013, Yang et al. published a systematic review on pelvic exenteration for rectal cancer . The 5-yPatients with a CUA also reported satisfactory control of voiding function, with a voiding style resembling that of patients with an ileal neobladder. Unfortunately, six patients required cystostomy because the sphincter urethra muscle was sacrificed due to probable cancerous invasion. These patients voided via an inserted catheter that was exchanged once a month, much like those with an ileal conduit. An obvious difference between neobladder and bladder-sparing surgery is that the neobladder is made using intestine, which results in inevitable long-term complications such as mucus production, nutritional abnormalities, metabolic acidosis, skeletal demineralization, and a risk of malignant transformation in the intestinal segment , 45. No In conclusion, this prospective study demonstrates the effectiveness of the ileal flap method for reducing CUA leakage and improving quality of life. Our results suggest that this new method should be evaluated using a similar trial design for patients with advanced rectal patients, to avoid colostomy.