Periodic mountain pine beetle outbreaks have killed millions of hectares of lodgepole pine forests in western North America. Within these forests some pine trees often remain alive. It has been rarely documented whether anatomical defenses differ between beetle-killed and remaining live pine trees, especially at the northern latitudinal range of beetles in North America. In this study, we compared the resin duct-based anatomical defenses and radial growth between beetle-killed and live residual lodgepole pine trees, and we characterized the resin ducts and the growth of the residual trees before and after outbreak. We found that tree radial growth was not associated with tree survival. The best two predictors of tree survival were resin duct size and production (number per year). Trees having larger but fewer resin ducts showed higher survival probability compared to those with smaller but more abundant resin ducts annually. Residual trees had larger resin ducts prior to the outbreak and continued having so after the outbreak. We further categorized residual trees as healthy (having no signs or symptoms of insect or pathogen attacks), declining (with signs or symptoms of biotic attacks), and survived (from mountain pine beetle attacks during the outbreak) to investigate resin duct-based anatomical defenses among them. Healthy trees had consistently larger resin ducts than declining trees in the past 20 years in post-outbreak stands. Survival trees ranked between healthy and declining trees. Overall, these results demonstrate that resin duct size of lodgepole pine trees can be an important component of tree defenses against mountain pine beetle attacks and suggest that lodgepole pine trees with large resin ducts are likely to show resistance to future bark beetle attacks. Dendroctonus ponderosae Hopkins, Coleoptera: Curculionidae) outbreaks had historically occurred in the western United States and Canada (British Columbia). Recently, however, outbreaks have shifted to higher elevations (Pinus contorta Douglas ex Loudon var. latifolia Engelm. ex S. Watson)  . Such ou Watson) . Some in Watson) . Althoug Watson) , the anaConifer trees have developed constitutive and inducible defense mechanisms that integrate both anatomical structures and toxic chemicals against bark beetle attacks . During Historically, studies on pine–bark beetle interactions have mainly focused on tree chemical defenses e.g., , while tMountain pine beetle outbreaks have altered forest stand conditions, including soil moisture, soil nutrients, soil microbial communities, and aboveground plant communities , and, inIn this study, we focused on the recent MPB outbreak in the beetle’s most northern range in the lodgepole pine forests of western Canada. We compared the growth rate and resin duct characteristics between beetle-killed and residual pine trees to determine the role of growth and defenses in tree survival under high intensity bark beetle attacks. We hypothesize that residual pine trees would have more or larger anatomical defense structures but lower radial growth compared to MPB-killed trees as residual trees might allocate more resources towards defense rather than growth. Furthermore, we investigated whether post-outbreak stand conditions altered the historical radial growth and resin duct defenses by the comparing growth rate and resin duct characteristics of residual pine trees between pre- and post-outbreak periods. Considering the reduced competition among overstory pine trees in the post-outbreak stands, we hypothesize that residual pine trees would produce more carbon resources, which may lead to a greater allocation to anatomical defense but can cause delayed growth release after MPB outbreak, compared to before outbreak. Finally, we tested whether radial growth and resin duct defenses varied among different categories of residual pine trees for the most recent 20 years of growth. We hypothesize that healthy residual pine trees would grow faster and have stronger anatomical defenses than trees showing symptoms of declining.Initially, we used provincial MPB mortality maps to select sites showing various levels of pine mortality in western Alberta . After tIn each plot, we randomly selected up to three MPB-killed and six residual (two from each of the three categories) trees from the dominant or codominant crown class. Since some plots did not have trees in all three residual tree categories, the number of trees selected in each category varied among plots. In total, we selected 140 beetle-killed and 210 residual  trees. We cored the selected residual trees once at DBH on the south aspect using a 12 mm increment borer in May 2016, and, thus, the last complete ring formed on the cores occurred in 2015. From beetle-killed trees, we took a single wedge at the same height and aspect as the residual trees. Each wedge contained at least 15 years of growth counting back from the year of death. In addition, we recorded the DBH of all residual pine trees sampled. However, we could not measure the DBH of all beetle-killed trees because their bark had fallen off over the years.™ . We measured the ring width (mm) from bark to pith on all samples by WinDendro™ . If the ™ .We developed a master chronology based on the ring width of the cores taken from 76 healthy residual trees. The master chronology was used to identify any missing or false rings on the cores before we assigned a particular ring to a calendar year. We determined the year of tree death by cross-dating using COFECHA . However-1]), total resin duct area (sum of resin duct area in a 9mm wide sampled area per year[mm2 yr-1]), resin duct size (mean size of resin ducts in a 9mm wide sampled area per year[mm2 yr-1]). We also standardized two resin duct characteristics to the sampled area as resin duct density  [no. mm-2 yr-1]) and relative resin duct area (percent area occupied by resin ducts per year within the ring area [% yr-1]) on each core or wedge.We used a fixed sampling width (9 mm) of each annual ring on each increment core or wedge to quantify annual resin duct characteristics using ImageJ : resin d-1) and basal area increment  to represent tree radial growth. Working under the assumption that tree rings are concentric circles, the BAI was calculated using tree radii and ring width data. Tree radius was calculated by dividing tree DBH by two. Since we could not measure the DBH of beetle-killed trees, we only incorporated the BAI of residual trees in our data analyses.We used ring width  and resin duct characteristics between residual and MPB-killed trees. We accepted the year of the earliest tree death caused by MPB as the year the outbreak started at each site.via linear mixed-effects models, using plots nested in sites as a random effect with R package lme4  on wedges counting back from the last year when a complete tree ring (latewood) was formed . We firsage lme4 . When we(lsmeans .lme4. The response variables for the models were the tree status (live or dead) and the explanatory variables included ring width and the five resin duct characteristics. All residual trees sampled were included in the analyses. We took the following five steps to select the best explanatory variables for model fitting.We used mixed effects logistic regression models to determine the probability of tree survival during MPB outbreak using R package vegan  to visualize differences in the resin duct characteristics and ring width among the four categories of trees sampled using R package vegan . The twovegan . This prHmisc  and that were not correlated to each other (the results of Step 2 above). This process yielded a total of 33 models Table 2.In Step 4, we used the Akaike Information Criterion (AIC) to select the optimal set of variables in each model. The model with lower AIC was considered to have the optimal set of variables . We conspROC , and the area under receiver operating curves (AUC). The models with a lower AIC and higher AUC usually performed better than the models with a higher AIC or/and lower AUC . The ∆AIpROC .To compare growth rate and resin duct characteristics before and after MPB outbreak, we divided cores into two sections as the pre-outbreak and the post-outbreak. The post-outbreak section was further divided into three overlapping periods: 3 years, 5 years, and the total (period from the first year after the outbreak until the last complete ring formation in 2015). Because the first year the outbreak started was different among sites, the total post-outbreak period was also different, ranging from 8 to 10 years.We first calculated the means of all resin duct characteristics, ring width, and BAI for each post-outbreak period. We then compared these means with the means of the same variables calculated from the 10-year pre-outbreak period under the first research question above. These comparisons were made for every category of residual trees separately and using linear mixed-effects models, with trees nested in plots and plots nested in sites as a random effect. We used DBH as a covariant to account for the influence of tree size. When we found differences, we used lsmeans to conduct pairwise post-hoc tests.We selected four overlapping periods on each increment core: 3-, 5-, 10-, and 20-year, counting back from the last complete ring formation in 2015. We then calculated the mean of each resin duct characteristic, ring width, and BAI for each period and compared their means among residual tree categories using linear mixed-effects models, with plots nested in sites as a random effect. We used tree DBH as a covariant to account for the influence of different tree sizes in these models. When we found differences, we used lsmeans to conduct pairwise post-hoc tests.Mean ring width was similar between beetle-killed and residual trees for all three pre-outbreak  periods Table 3.Overall, resin duct characteristics were more important for estimating the survival probability of pine trees than ring width in models. Models classified residual trees as better than killed trees (82–88% vs. 36–80% respectively). Models including both resin duct production and resin duct size had a lower AIC (< 320) and higher AUC (≥ 0.89) and performed better in classifying killed and residual trees (67-80% vs. 83-88% respectively), compared to the models that included only one of the two resin duct characteristics or none  Table 2.Both declining and survived trees showed reduced ring width during the first 5 years after the outbreak as compared to before the outbreak Figure 3In general, almost all resin duct characteristics in all residual trees had a higher value in the post-outbreak growth periods as compared to the pre-outbreak growth period Figure 4As a covariant, DBH was significant in models that compared resin duct production of healthy trees between the 3-year post-outbreak and the pre-outbreak periods, total resin duct area of healthy trees between the 3-year post-outbreak and the pre-outbreak periods, and total resin duct area of healthy trees between 5-year post-outbreak and pre-outbreak periods.We found no differences in ring width among the three categories of residual trees for the recent 3-, 5-, 10- and 20-year growth periods. By contrast, BAI varied among residual trees Figure 5Among all five resin duct characteristics, only resin duct size Figure 6Through this study, we showed that resin duct characteristics can be critical components of pine survival before and after MPB outbreaks. Overall, beetle-killed trees had smaller resin ducts than residual trees and residual trees continued to have larger resin ducts after MPB outbreak. By increasing the volume of resin flow, larger resin ducts likely substantially reduced the probability of successful beetle colonization on residual trees by providing sticky physical barriers, sealing beetle entry wounds, and releasing toxic compounds . Thus, tAmong the anatomical characteristics measured, the number of resin ducts did not seem to be as important as their size; lodgepole pine trees with relatively smaller but more resin ducts were killed during MPB outbreak, and trees with larger but fewer resin ducts had a higher probability of survival during outbreak, suggesting a possible trade-off between size and number of resin ducts in pine trees . In addiFirst, larger resin ducts likely result in the storage and biosynthesis of a higher volume of resin, thereby increasing resin accumulation within the tree . ConsequOur results emphasized the importance of resin duct size over the number of resin ducts. These results are partially in disagreement with former studies that reported a higher number of resin ducts in resistant trees . Above, We consistently found enhanced anatomical defense structures in residual pine trees during post-outbreak as compared to pre-outbreak periods. This is the first report of enhanced anatomical defenses in pines after bark beetle outbreaks. Two possible post-outbreak stand conditions may explain these results. First, these stands may not provide optimal conditions for tree growth following MPB outbreaks . In factOverall ring width was similar between beetle-killed and residual trees and was not associated with pine survival in the best predictive models. However, ring width was correlated with tree size, suggesting that, with the same ring width, larger trees can have more annual BAI compared to smaller trees. Therefore, DBH should have been used as a covariant to account for the influence of tree size in our models. However, since we could not accurately measure the DBH of all beetle-killed trees, this created some potential problems for data analysis and interpretation. To resolve this issue, we used the DBH of beetle-killed trees with intact bark for further comparison with the DBH of residual pine trees. These results showed that beetle killed trees had larger DBH than residual trees . The rolIn Alberta, MPB outbreaks happened unexpectedly as a large number of beetles dispersed from British Columbia and landed in the forests in the west of Alberta. In contrast to the historical range of MPB, Alberta did not experience the regular population cycles of beetles  . AlthougIn conclusion, we showed that anatomical defense structures can be critical components of lodgepole pine survival during MPB outbreaks. Furthermore, the same defense structures have likely continued playing major roles in defending residual trees against biotic attacks in post-outbreak stands. Among anatomical defense structures investigated, resin duct size appears to be an important indicator of tree resistance to bark beetles. However, the literature on the resin duct characteristics of conifers shows variations, but underlying factors for such differences are not clear and require further investigations. It is not clear whether resin duct size is heritable, but, if so, we expect that the next generation of lodgepole pine forests in western Alberta would be more resistant to future MPB attacks. Considering the climate change enhanced bark beetle outbreaks in North America , we suggThe datasets generated for this study are available on request to the corresponding author.SZ and NE conceived and designed the experiments. SZ performed the experiments. SZ and NE performed statistical analysis and generated tables and figures. SZ wrote the manuscript. NE contributed to editing.Funding for this research was provided by fRI Research, Alberta Agriculture and Forestry, Alberta Innovates-BioSolutions, Forest Resource Improvement Association of Alberta, and NSERC–Discovery Award to NE. 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.


Ablepharon macrostomia syndrome (AMS) is a rare congenital malformation disorder caused by the autosomal-dominant mutations in gene TWIST2. Patients affected by the disease present abnormalities in ectoderm-derived structures mainly consisting in major facial dysmorphic features and rarely in visceral anomalies. The only laryngo-tracheal defect reported is malacia, with no reference to any anatomical stenosis. We describe a unique case of laryngo-tracheal stenosis in a woman, with genetically confirmed AMS currently followed at our Department.A 37-year-old Caucasian woman was admitted to the intensive care unit for acute dyspnea that required orotracheal intubation followed by tracheostomy.The bronchoscopy revealed abnormal tracheal tissue at the level of the cricoid and the first three tracheal rings reducing airway caliber by 80% (grade III according to the Cotton-Meyer classification). Treatment of the stenosis by means of temporary tracheostomy and corticosteroids therapy resulted in airway patency restoration and patient’s return to her normal activities. Bronchoscopy at four and five months showed disappearance of the abnormal tissue and a residual anatomical laryngo-tracheal stenosis of about 20% (grade I according to the Cotton-Meyer classification) of the normal airway caliber.To our knowledge, this is the first patient affected by AMS presenting with laryngo-tracheal stenosis. Typical congenital malformations in ectoderm-derived structures, mainly in the face, characterize ablepharon macrostomia syndrome (AMS). They include: absent or hypoplastic eyelids, fusion defect of the mouth, abnormal ears, skin defects, normal intellectual and motor development, variable abnormalities of the nipples, genitalia, fingers, and hands, and poor growth . AMS wasA 37yo Caucasian woman was seen in May 2018 at our Department of Thoracic Surgery for major dyspnea on exertion. She was known for AMS with the specific genetic mutation in the gene TWIST2 (c.223G > A), no pathogenic sequence changes were found in both her parents . The patThe patient underwent 17 previously maxillofacial operations requiring intubation, including multiple surgeries for eyelids and ears lobes reconstruction, mastoplasty for mammary glands absence and lips correction. The last procedure had been performed ten years before the onset of symptoms. In her lifetime, the patient never complained dyspnea and/or shortness of breath, although she reported a quiet lifestyle.At the time of our evaluation, she reported dyspnea on exertion in the previous 18 months with an acute episode that occurred six months before. The acute dyspnea episode caused intensive care unit (ICU) admission, with orotracheal intubation by means of a 5 mm tube, subsequent tracheostomy and prolonged mechanical ventilation. Flexible ICU bronchoscopy showed endotracheal tissue approximately at the cricoid level and the first three tracheal rings resulting in an airway stenosis of 80% (grade III according to the Cotton-Meyer classification), compatible with inflammatory edema of the mucosa, above the tracheostomy site. The distal trachea was normal.She received corticosteroid obtaining a resolution of symptoms and a return to spontaneous breathing. Tracheostomy cannula was removed after 19 days and the patient was discharged from the hospital.The patient returned to her normal daily activities. Thereafter, she continued to complain of dyspnea for intense efforts, despite the improvement in the symptoms. She was then referred to our Department for reevaluation. Laryngoscopy showed a normal glottis. A flexible bronchoscopy performed at the beginning of June 2018 revealed a circumferential airway stenosis involving the cricoid cartilage and the first tracheal ring, resulting in an airway stenosis of 50% (grade I according to the Cotton-Meyer classification) Fig. .Fig. 1EAMS is an infrequently reported congenital malformation disorder caused by mutations in the TWIST2 gene causing defects in ectoderm-derived structures . AbnormaLaryngo-tracheal anomalies do not belong to the AMS phenotype  but mighIn light of the patient’s history, we hypothesize that the laryngo-tracheal stenosis has been always present in this woman, although primary etiology cannot still be recognized. To our knowledge, this is the first case of laryngo-tracheal stenosis in AMS reported in the literature. Further considerations are necessary after future patient evaluation.


Equus hemionus onager) in local communities in southern Iran. We asked about locals’ perceptions of six potential management strategies intended to lessen human-onager conflict. We found that human-onager conflict was restricted to 45% of respondents within the Bahram-e-Goor Protected Area, all of whom were involved in farming or herding activities. Locals within the protected area were more knowledgeable about onagers and valued onagers more than those living outside the protected area. The perceived value of onagers increased with level of education, total annual income, and perceptions of onager population trends; the perceived value of onagers decreased with the magnitude of conflict between onagers and locals. To tolerate or avoid conflicts with onagers, locals were supportive of monetary compensation and changing from a traditional lifestyle to industrialized farming (for farmers) or livestock production (for herders) with the help of government; locals did not support selling land to the government. Our study is among the first in human-wildlife conflict and local attitudes towards an endangered species and its recovery in Iran. We conclude that current levels of human-onager conflict are relatively low and perceived value of onagers is still relatively high. Therefore, wildlife authorities should consider the development of mitigation strategies with local communities before conflicts intensify.Human-wildlife conflicts restrict conservation efforts, especially for wide-ranging animals whose home ranges overlap with human activities. We conducted a study to understand conflicts with, and factors influencing the perceived value of an expanding population of onagers ( Increasingly, wide-ranging animals share landscapes with humans and their livestock, triggering human-wildlife conflict which can impart harm on both sides –3. HumanConservation efforts aimed at reducing human-wildlife conflict have been successful in bolstering population sizes and expanding geographic ranges for many of species . NeverthThe black arrow shows a negative effect of conservation on human livelihoods, which can reduce or negate conservation efforts. A destructive loop is created when conservation success results in increased conflict levels, which results in a feedback of retaliatory killings and renewed conservation efforts. Although conflict escalation leads to conservation failure, conflict mitigation breaks the feedback loop and leads to conservation success.Positive attitudes toward wildlife are a powerful driver for wildlife recovery . The mosEquus hemionus) is found throughout deserts and desert-steppes of Central Asia. With an estimated global population of 55,000, the species is still relatively abundant in some areas, but it occupies less than 3% of its historic range [E. h. onager) is an endangered subspecies of Asiatic wild ass [The Asiatic wild ass . . Equus hwild ass . OnagersThe mechanisms of onager population suppression within BPA are not well understood , but poaWe sought to understand factors contributing to human-onager conflicts in rural communities in and around BPA. We used structured questionnaires to understand the nature and extent of the onager-human conflict, quantify local attitudes and knowledge, and assess support for potential management strategies. We hypothesized that (1) people residing within BPA would experience higher levels of conflict compared to those outside BPA; and (2) the occurrence and magnitude of conflicts would predict both people’s attitudes towards onagers, and their preferences for management strategies. Our study provides the first quantitative data on human-onager interactions to guide future recovery strategies for this endangered subspecies.Participants gave their consent verbally after being explained about the aim of the survey, topic of the different sections on the questionnaire, and how the interview would proceed. Consent was obtained by having the participants state that they agreed to participate. The data were collected and analyzed anonymously. The project, including the questionnaire and its consent acquisition procedure, received the approval from the Iranian Department of Environment (permission number: 33183/92 issued at 9 Dec 2013).2 BPA and in a surrounding 50-kilometer buffer  and Turk terebinth pistache (Pistacia atlantica).We conducted our study in the Fars, Kerman, and Yazd Provinces on the central plateau of Iran. We were especially interested in differences between locals from the 3,747 kmGazella bennettii), wild sheep , wild goat (Capra aegagrus), and Asian houbara bustard (Chlamydotis maqueenii). Additionally, BPA is home to wild boars (Sus scrofa) and gray wolves (Canis lupus), with the former regarded as a threat to crops [Ovis aries and goat Capra hircus, occasionally camel Camelus dromedarius) per square kilometer are permitted to graze inside the BPA . Frequent droughts are the main impediment to farming in the area, which have led to a transition from water-intensive crops such as wheat to drought-resistant orchards and livestock .In 1972, BPA was established to protect onagers and other threatened species, including Indian gazelle  respondents within BPA and 11 (7%) outside BPA were unaware of onagers, resulting in a sample size of 100 interviews from within BPA and 142 from outside BPA . We adopWithin BPA, we interviewed an additional 101 farmers and herders  to quantify levels of agreement toward six strategies to reduce human-onager conflict.To ensure that our questionnaires addressed the objectives of our study, we conducted 25 pilot interviews with locals where we explored their views about their livelihoods, BPA, onagers, and other wildlife. We subsequently used that information to modify a pre-existing questionnaire used to assess public attitudes toward Asiatic wild ass in Mongolia . To initWe used responses from sections #1–3 as predictors for answers to questions in section #4 , and in section #5 .We hypothesized that socio-economic background of locals would influence both the perceived value of onagers and acceptability of potential management strategies. We asked locals about their gender, age, and level of education . We recorded livestock ownership (the number of livestock) for herders and farm size for farmers. We asked locals to categorize their total annual income in two categories of <$5000USD and >$5000 USD.We hypothesized that conflict with onagers would influence both the perceived value of onagers and acceptability of potential management strategies. To assess the relative magnitude of human-onager conflict, we asked locals to rate the degree of conflict with onagers from no conflict to severe conflict coded from one to five. We asked locals about the occurrence, type, and timing of conflicts with onagers.We hypothesized that knowledge of locals would influence the perceived value of onagers. To quantify local knowledge about onagers, we asked if four incorrect statements about onager biology were true or false. When respondents stated that incorrect statements were correct, we recorded answers as a “0”. When respondents stated that incorrect statements were incorrect, we recorded answers as a “1”. We averaged these four answers to generate a knowledge score about onagers ranging from zero to one, indicating low and high levels of knowledge, respectively. Additionally, we asked about perceived population trends of onagers ; we predicted that those who thought onager population had been decreasing would value the species more.To quantify the perceived value of onagers, we asked locals to rank agreement with fifteen statements from “strongly disagree” (coded as 1) to “strongly agree” (coded as 5). We averaged answers to these fifteen statements to produce a semi-continuous “value score” ranging from one to five , S1 Fig.We inquired about the acceptability of six strategies aimed at reducing human-onager conflict, targeting only individuals within BPA . We deliberately excluded fencing because we were most interested in management strategies that would not further fragment the distribution of onagers. The strategies were: (1) selling land to the government; (2) exchanging 50% farmland/pastureland within BPA for an equivalent amount of land outside BPA; (3) changing from a traditional farming/herding lifestyle to industrialized farming (for farmers) or livestock production (for herders) with the help of government; (4) accepting monetary compensation to tolerate onager conflict; (5) accepting a sedentary lifestyle instead of a nomadic one; and (6) supplementary feeding of livestock for a period in the year with the help of the government. The last two strategies were asked only from herders (who are nomadic within BPA) and not farmers (who are sedentary within BPA). We recorded responses in three categories: disagree, neutral, and agree. Unlike previous questions, we restricted responses to three categories because 5 categories were challenging for locals to understand.2) and Wilcoxon signed-ranks tests.We used R software  for statistical analysis. To compare responses within and outside BPA, we used Chi-square (χUsing beta regression (Betareg package in R ), we rel2 tests to select models and used R2 and a concordance-index (c-index) as a measure of predictive performance [To predict factors influencing acceptability of management strategies, we related socio-economic predictors  and level of conflict with onagers to three response categories of disagree, neutral, and agree for each strategy  using ordinal logistic regression models (lrm function in package rms ). Due toformance .Human-onager conflict was restricted to respondents involved in farming or herding within BPA . No confForty-five farmers and herders within BPA experienced conflict with onagers, categorizing it as little (31%), some (20%), high (20%), or severe (29%). Conflicts with onagers were largely a reoccurring issue spanning multiple years . The proportion of all human-wildlife conflict attributed to onagers ranged between 1% and 100% (median = 30%). Most conflict with onagers were with respect to crop depredation, and occurred mostly in summer (58%), least in winter (20%), and at intermediate frequency in fall (40%) and spring (30%); 4% of respondents claiming that conflicts with onagers occurred year-round. The crops most susceptible to depredation were alfalfa (84%), wheat and barley (44%), fruit orchards and vegetables (22%), and corn (9%). Only three respondents (7%) reported conflicts over pasture.Knowledge about onagers was poor overall. Locals within BPA were more knowledgeable  and had higher knowledge scores . Locals’ level of education, total annual income, and perceived onager population trends affected perceived value of onagers positively. Level of conflict with onagers negatively influenced the perceived value of onager, although explanatory power of the model was low  or livestock production (for herders) with the help of government. Farmers and herders agreed least with selling land to the government. Farmers and herders were ambivalent towards exchanging 50% farmland/pastureland within BPA for land outside BPA and herders towards accepting a sedentary lifestyle instead of a nomadic one or supplementary feeding of livestock for a period in the year with the help of the government .Locals reporting high levels of conflict with onagers were less likely to agree with exchanging 50% of farmland/pastureland within BPA for land outside BPA, and were more likely to agree with monetary compensation to tolerate conflicts with onagers . Age, liAcinonyx jubatus venaticus) and Persian leopard (Panthera pardus saxicolor) [Our study is among the first to assess attitudes of local people toward human-wildlife conflict  and recoxicolor) ,40. Howexicolor) . Althougxicolor) ,43.Although conflicts were restricted exclusively to rural respondents within BPA, these individuals did not value onagers less than respondents outside BPA. This lack of difference suggests that human-onager conflict is either low, or that even severe and reoccurring conflict with onagers has not yet lead to overly-negative attitudes . AdditioFarmers and herders seem willing to switch from a traditional farming/herding lifestyle to a more industrialized form of crop and livestock production. If managed well, this could reduce the need for land conversion and minimize competition for pasture between wild and domestic ungulates. One potential drawback of industrialized farming might be increased human presence and transportation around onager habitats. However, the effects of such changes need to be carefully evaluated and local people and conservationists must come to a common understanding of the socio-economic, cultural, and ecological consequences of such changes. Selling or exchanging land from inside BPA for land outside BPA, on the other hand, received little support. Although separating wildlife and people can be highly effective in reducing conflict, forcing people to leave can escalate human-wildlife conflict ,51.Both of the most supported strategies  or livestock production (for herders) with the help of government) entail financial incentives. Critically, locals were less supportive of accepting a sedentary lifestyle instead of a nomadic one when financial incentives were not involved. So, satisfying the economic needs (or desires) of local communities may assist in reaching a common solution in human-wildlife conflicts ,47.Given the localized nature of human-onager conflict, monetary compensation through insurance schemes may be the most cost-efficient means of conflict reduction. Insurance schemes do not require government support necessarily. However, insurance schemes additionally require that locals protect their farms to be eligible for coverage offered by insurance . Advice on fence design and affordable technologies, like solar-powered electric fencing, could help reduce conflicts by both onagers and wild boars .(DOCX)Click here for additional data file.S2 TableCronbach’s alpha consistency analysis is used to generate Cronbach’s alpha score, which indicates high consistency between components of the value score.(DOCX)Click here for additional data file.S3 TableN questionnaires represent the total number of interviews (regardless of respondents’ familiarity with onagers).(DOCX)Click here for additional data file.S1 FigWe asked locals to rank agreement with fifteen statements from “strongly disagree” (coded as 1) to “strongly agree” (coded as 5).(DOCX)Click here for additional data file.S1 Text(Farsi version of the questionnaire was used in interviews).(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.


The primary goal of this study was to create a frequency map of a series of surgically treated both-column fractures and to explore its implications on surgical management.We used a consecutive series of 71 both-column fractures to create 3-dimensional reconstruction images, which were superimposed and oriented to fit a model hemipelvis template by aligning specific pelvis landmarks. Fracture lines were identified and traced to create a fracture map of both-column fractures. Then the possible clinical implications of fracture line map were explored.n = 65, 92%) formed a Y-shaped pattern, which highlighted the consistency of the patterns.Fracture location is closely related to the distribution of fracture line. Of 71 fractures that met the criteria for inclusion, we found the most common pattern demonstrated by coexisting fracture lines. The anterior column was involved by 66% of the fractures extending obliquely from the anterior superior spine to the ischial spine, while 62% of the fractures involved the anterior column extending approximately vertically from the iliac crest to the acetabular roof. Additionally, 39% of the fractures involved the posterior column traversing posterior wall. Furthermore, the high fracture line intensity (Surgically treated both-column fractures display very common patterns. The most common pattern is the low anterior column fracture in nearly two thirds of cases, the high anterior column fracture in three fifths of cases and the posterior column fracture with posterior wall involvement in nearly two fifths of cases. These study results may help surgeons to yield insight relevant to surgical approaches, reduction, fixation strategies and even implant design for both-column fractures. Both-column fractures typically result from high energy trauma, which are complex injuries with technically demanding fracture patterns. In these fractures, the posterior column is usually detached and displaced, possibly with a posterior wall fracture . A seconIn our clinical practice, we have found that associated both-column fractures are frequently associated with posterior wall fractures. However, when posterior wall fractures affect the clinical outcome of both-column fractures, the best way to manage such injuries has not been clearly defined. Many classification systems for acetabular fractures have been established, which may sometimes differ from clinical data sets of actual fracture patterns. A full understanding of the fracture patterns is helpful to develop appropriate surgical strategies. It has been reported that three-dimensional computed tomography 3DCT) reconstruction technology can better understand the three-dimensional structure of acetabular fractures and be used as a guide for surgical planning DCT recon. The purAt our level I trauma center, a search in the administrative database showed three hundred and seventy two patients with acetabular or pelvic fractures were admitted into the trauma department from January 2009 to January 2018. Inclusion criteria were (1) age of 18 years or older, (2) acute both-column fracture, (3) complete radiographic assessment including X-ray and CT scan. Exclusion criteria included: (1) anatomical details obscured by artifacts, (2) severe comminuted fractures which were difficult to determine fracture lines; (3) poor quality CT data or lack of complete radiographic images. 301 patients were excluded. The remaining 71 patients were analyzed in this study. All radiographs, CT scan and surgical reports were evaluated by two authors to establish the diagnosis.This study was approved by the Ethics Committee and Institutional Review Board of West China Hospital. All investigations were conducted in conformity with ethical principles of research. The ethics committee waived the need for consent and patients provided verbal consent to participate in the study.Referring to the mapping method that Cole et al.  and ArmiThen these views were imported into Macromedia Fireworks MX software  to overlap and orient fracture patterns onto a template hemipelvis image. Proper rotation and normalization were guided by aligning specific pelvic landmarks: greater sciatic notch, ischial spine and ischial tuberosity. Once proper anatomical alignment was obtained, fracture lines were identified and were traced on the back of the pelvis Fig. . At the In addition, zones were defined on the basis of pelvic anatomy and/or key muscular origins and insertions Fig. a. Based Quality of reduction was estimated on anteroposterior (AP) pelvis and Judet views that taken immediately after surgery by Matta’s criteria : anatomiA total of 71 patients met the inclusion criteria, among which were 43 male patients and 28 female patients, with an average age of 41 years. All patients underwent surgery. The most common injury mechanism was fall from height  had high anterior column fractures, 47 cases (66%) had low anterior column fractures, twenty-eight cases (39%) had posterior column fractures involving the posterior wall  the fracture line from the point near the anterior superior spine towards the ischial spine, (2) that from the iliac crest to the acetabular roof, and (3) that traversed posterior wall. Furthermore, in the cases of both-column fractures we studied, posterior wall fractures tended to be concomitant. The most common pattern was a fracture line traversing posterior wall.These results can facilitate the identification of critical locations at which to access displaced fractures. Surgeons can apply our study results to guide the placement of implants for optimal screw purchase and internal fixation. Additionally, we can also combine anatomical parameters of the pelvis with the main fracture lines to design a novel internal fixation device, which is the focus of further research in the future.Our study has several limitations. First, it was a retrospective review of prospectively gathered data for a small number of patients. Second, our analysis did not take potential variability in anatomy and injury mechanism into account. Third, some hemipelvis images did not match the hemipelvis model perfectly. Finally, fracture lines drawn on the hemipelvis model could slightly differ from true fracture morphology.In conclusion, the fracture maps of both-column fractures derived from 3D CT can aid in guiding the treatment of such complex fractures, in terms of surgical approaches, preoperative planning, and implant strategy.


Arabidopsis defense, which decreased the reproductive performance of one of the subsequent herbivores. The expression analysis of several defense-related genes demonstrated that the individual impact of mite and aphid feeding spread systematically, engaging the salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Interestingly, aphids feeding on the systemic leaf of the plant simultaneously attacked by mites, efficiently reduced the magnitude of the SA and JA activation, whereas mites feeding remotely increased the aphid-induced SA marker gene expression, while the JA-dependent response was completely abolished. We also indicated that the weaker performance of mites and aphids in double infestation essays might be attributed to aliphatic glucosinolates. Our report is the first to provide molecular data on signaling cross-talk when representatives of two distinct taxonomical classes within the phylum Arthropoda co-infest the same plant.In natural and agricultural conditions, plants are attacked by a community of herbivores, including aphids and mites. The green peach aphid and the two-spotted spider mite, both economically important pests, may share the same plant. Therefore, an important question arises as to how plants integrate signals induced by dual herbivore attack into the optimal defensive response. We showed that regardless of which attacker was first, 24 h of infestation allowed for efficient priming of the  In natural and agricultural conditions, plants are often attacked by multiple herbivores. Similar to single infestations, plant’s defense responses can modulate diverse pest performance with a broad to narrow specificity spectrum ,5,6,7,8.Arabidopsis leaves, a huge defense signaling gene expression reprograming was shown compared to that in non-infested control leaves from the same plant  spreads to non-infested, distal organs of the plant . Consequme plant . SimilarMyzus persicae Sulzer, 1776; Hemiptera: Aphididae) and the two-spotted spider mite, TSSM  are economically important arthropod herbivorous pests attacking a wide range of host plants and inflicting serious damage and crop yield losses. They may either attack the plant individually or share the same plant. Although mites and aphids are piercing-sucking herbivores, they suck up distinctly from the host-plant. TSSM feeds mostly from the cells of mesophyll tissues . As a control, mite-infested plants with empty cages clipped to the respective leaves were used [Tu+/Mp−]. TSSM reproductive performance—expressed as their oviposition rate—was measured in the number of mite eggs laid by all females on the Arabidopsis rosette. Aphids  were clipped on one leaf of the Arabidopsis (n = 6) was caged with 60 GPA females for 24 h. Then, aphids were manually removed from the plants and each plant was infested with 50 TSSM for 24 h [Mp+/Tu+ seq]. As a control, mite-infested plants with empty cages attached to the appropriate non-infested leaves were used [Mp−/Tu+]. TSSM reproductive performance was assessed on previously GPA-infested Arabidopsis plants and expressed as mite eggs laid by all females per an Arabidopsis rosette. To assess whether TSSM can activate defense against GPA and vice versa, two types of experiments were carried out. In the first one, one leaf of each Arabidopsis plants (n = 9) were infested with 50 TSSM females per plant. Mites fed freely on the leaves. After 24 h, all mite females were manually removed using a fine paintbrush and newborn PGA nymphs (5 per plant) were caged to one of the previously mite-infested leaves [Tu+/Mp+ seq]. Plants uninfested by mites, with newborn aphid nymphs caged [Tu−/Mp+] to appropriate leaves, were used as a control. After two weeks, the reproductive GPA performance on previously TSSM-infested and control (TSSM-uninfested) leaves was assessed and expressed as the total number of aphids per plant. In the second experiment, Leaf damage was evaluated using the leaves from the above-described experiment on GPA/TSSM sequential infestation. Trypan Blue (TB) staining was done according to . Mite-inRNA isolation from herbivore-infested and uninfested leaves was performed using GeneMATRIX Universal RNA/miRNA Purification Kit . Reverse transcription was performed using QuantiTect Reverse Transcription Kit .TM System  and the QuantiTect SYBR Green PCR Kit  with primers listed in ACT2 as an internal reference, according to the ΔΔCt method [For the analysis of transcript levels, we used the Bio-Rad CFX96 Toucht method . Signifit method .The statistical significance of the difference between means was assessed using the two-tailed Student’s test . Arthropoda co-infest the same plant. The presented data suggest that the role of SA and JA in the integration of signals triggering host-plant response to a combined attack might be less important than could be inferred from a single infestation, which gives room for other signaling mechanisms and more subtle regulations to work.This report is the first to show the signaling cross-talk when representatives of two distinct taxonomical classes within the phylum


We invert postseismic geodetic data to reveal subduction zone mechanical properties following a megathrust earthquake in Chile. Mw 8.8 Maule earthquake occurred close to the Chilean coast within a dense network of continuously recording Global Positioning System stations, which provide a comprehensive history of surface strain. We use these data to assemble a detailed picture of a structurally controlled megathrust fault frictional patchwork and the three-dimensional rheological and time-dependent viscosity structure of the lower crust and upper mantle, all of which control the relative importance of afterslip and viscoelastic relaxation during postseismic deformation. These results enhance our understanding of subduction dynamics including the interplay of localized and distributed deformation during the subduction zone earthquake cycle.Deformation associated with plate convergence at subduction zones is accommodated by a complex system involving fault slip and viscoelastic flow. These processes have proven difficult to disentangle. The 2010  The largest earthquakes occur on the gently dipping fault planes that comprise the shallow portions of subduction zones at convergent tectonic plate margins (Mw (moment magnitude) ≥8.5] subduction earthquakes have occurred in the era of satellite geodesy: the 2004 Mw 9.2 Sumatra-Andaman and the 2005 Mw 8.6 Nias-Simeulue, both in Sumatra, Indonesia; the 2010 Mw 8.8 Maule, Chile; and the 2011 Mw 9.0 Tohoku-Oki, Japan events (Mw ≥8.0) earthquakes have yielded useful, yet often conflicting conclusions regarding the inferred rheological properties. This is due to the difficulty of resolving the relative contributions of the various postseismic deformation mechanisms  earthquakes  matches the observed horizontal and vertical displacement magnitudes and azimuths, although we slightly underestimate total displacements in a few scattered locations . We reprThe model also explains the temporal evolution of postseismic deformation  just west of the high Andes. CM strain concentrates directly beneath the volcanic arc. Together, the OM and CM are responsible for the broad vertical deformation signal and change from near-field subsidence to uplift across the high Andes.MW flow results in a vertical deformation pattern characterized by uplift extending from the coastline to the volcanic arc with a transition to subsidence across the Chile-Argentina border and further east. This pattern contributes significantly to the total horizontal displacement field particularly in central Chile across-strike from the earthquake. As with the CM, MW strain concentrates beneath the volcanic arc. In contrast, LC flow minimally affects the postseismic deformation field contributing only about a centimeter of horizontal and vertical motion. The decrease in CGPS site density moving inland combined with the smaller size of the associated volumes affects our ability to resolve LC strain. However, those that are well resolved indicate relatively small amounts of distributed lower crustal strain , σn is the effective normal stress, ΔCFS is the coseismic Coulomb stress change on the creeping fault element, and V is the steady-state sliding velocity σn to be estimated in locations where afterslip occurs (fig. S7). In this framework, if a − b > 0, then the material is considered VS, and primarily stable sliding  occurs. VS regions inhibit earthquake nucleation and rupture propagation. In contrast, if a − b < 0, then the material is velocity weakening (VW) and potentially seismic . Although VW is required for earthquake nucleation, this condition is insufficient to fully predict slip behavior because a VW patch can be stable , exhibit complex characteristics including slow-slip and very low-frequency events, and participate in coseismic slip that initiates elsewhere. We refer to these regions as stable weakening. In contrast, we refer to fault patches that are both VW and unstable  as unstable weakening (UW).To explore megathrust fault frictional properties, we assume that the temporal evolution of afterslip is controlled by steady-state rate-and-state frictionOur frictional property estimates  are compThese results permit a more in-depth comparison with geological and geophysical evidence pointing toward structural control of Maule coseismic rupture. Despite the initial suggestion that coseismic slip was confined to the area between the Mocha Fracture Zone in the south and the Juan Fernández Ridge in the north  and 4 3, composeTherefore, significant shallow afterslip was confined to a portion of the plate interface underlying the AP, and the frictional behavior is controlled by the hanging wall rheology . Near-trThe shallow megathrust region beneath the AP is often assumed VS, thus impeding dynamic rupture . The up-0 and ∆τ(t) are the background stress and postseismic stress change and 17 to 1018 Pa s, which rapidly increase during the first few weeks after the earthquake to steady-state values of 1019 to 1020 Pa s. This transient behavior lends support to the notion that a bi-viscous Burgers rheological model  forearc MW, a hot  LC below the volcanic arc, and a warm backarc  (44). ThThe strong, cold, and wet MW corner points toward the presence of fluid- and antigorite-enriched, semibrittle serpentinite . The spaQ; The contrast in steady-state viscosity across the subducting slab at depths <150 km and the relatively high OM viscosity, in particular, is consistent with estimates derived from postseismic observations following the 1960 Chile, 2011 Tohoku-Oki, and 2012 Indian Ocean earthquakes Q;  .The dense regional geodetic network across the Central Chile subduction zone and associated long time series of surface deformation allow us to explore how afterslip and viscoelastic relaxation deform the lithosphere-asthenosphere system following a giant earthquake. These data, when combined with an innovative inversion approach, reveal lateral variations in frictional properties and a first-order thermal structure that controls the strength of the oceanic and continental lithosphere-asthenosphere system, furthering the notion that geodetic imaging is an important tool for determining the mechanical properties of subduction zones and better understanding the development of seismic hazard, complementing more traditional techniques such as seismic tomography.


Their findings implicate an extensive yet understudied role for retroviruses in shaping the evolution of placental gene regulatory networks.The co-option of endogenous retroviruses (ERVs) is increasingly recognized as a recurrent theme in placental biology, which has far-reaching implications for our understanding of mammalian evolution and reproductive health. Most research in this area has focused on ERV-derived proteins, which have been repeatedly co-opted to promote cell–cell fusion and immune modulation in the placenta. ERVs also harbor regulatory sequences that can potentially control placental gene expression, but there has been limited evidence to support this role. In a recent study, Dunn-Fletcher and colleagues discover a striking example of an ERV-derived enhancer element that has been co-opted to regulate a gene important for human pregnancy. Using genomic and experimental approaches, they firmly establish that a primate-specific ERV functions as a placenta-specific enhancer for  The ancestral form of our placenta emerged roughly 130 million years ago, marking a key evolutionary innovation that enabled live birth in mammals [One of the unique challenges to studying the placenta is the fact that it exhibits unexpectedly wide variation in form and function across species, despite performing a conserved role supporting fetal development. The mammalian placenta shows great diversity across species in its morphology and tissue organization, mechanisms of implantation and invasion, and physiological regulation ,3. Even What fueled the explosive evolutionary diversification of the mammalian placenta? Addressing this question is crucial for understanding mammalian reproductive biology and human-specific conditions of pregnancy ,8. IntriRetroviruses, which include contemporary viruses such as human immunodeficiency virus (HIV) and human T-lymphotropic virus (HTLV), have been infecting vertebrates for over 450 million years . A key sSyncytin-1, which originates from a retroviral envelope gene. In primates, Syncytin-1 was repurposed for the development of a multinucleate tissue layer known as the syncytiotrophoblast, which separates maternal and fetal bloodstreams in the placenta [Syncytin-like retroviral proteins have been reported to be expressed in the placentas of nearly all mammals, yet Syncytins in different lineages derive from at least 10 independent infections by unrelated retroviruses [Since the discovery of ERVs decades ago, a number of retroviral proteins have been identified that have been co-opted to perform a wide range of biological functions . Notablyplacenta . Remarkaoviruses . These foviruses .Functional studies have revealed that ERV-derived proteins tend to be co-opted for three roles in the placenta: mediating cell–cell fusion to form a multinucleate barrier, suppressing maternal immunity, and protecting the fetus from exogenous viruses ,16. HoweThe idea that repetitive elements such as ERVs may serve as basic components of gene regulatory networks can be traced back over a half-century to the foundational work of Barbara McClintock, Eric Davidson, and Roy Britten ,19. ERVsWhile genomic studies point to ERVs as an abundant source of placenta-specific regulatory elements, their biological relevance to pregnancy remains largely unexplored. Given their retroviral origins, ERVs are not expected to have any benefit for the host, and some may even exhibit pathological activity. Therefore, without further experimental support, it remains unclear whether ERV-derived regulatory activity is actually relevant for placental function.PLOS Biology [corticotropin-releasing hormone (CRH), which encodes a peptide hormone produced in massive quantities in the human placenta. Placental CRH levels undergo exponential increases throughout gestation, and misregulation of CRH is associated with premature and post-term birth [CRH is regulated is important for understanding the molecular control of birth timing but is complicated by the fact that placental expression of CRH is unique to primates. In all other mammals, CRH is expressed primarily in the hypothalamus and the brain [In a study published in this issue of  Biology , Dunn-Flrm birth . Determihe brain .CRH is determined by primate-specific regulatory sequence near the gene. Through their examination of the noncoding genomic region surrounding human CRH, they identify a primate-specific ERV insertion located 2 kilobase pairs upstream of the gene. The authors find little transcriptomic evidence for spliced CRH transcripts originating from the ERV, which suggests that the element may act as an enhancer rather than a promoter. The insertion is an LTR sequence that originates from an ancient retrovirus named transposon-like human element 1B (THE1B), which invaded the anthropoid primate lineage approximately 50 million years ago and dispersed roughly 20,000 elements in the genome before eventually becoming inactivated by mutations. On a genome-wide level, genes located near THE1B-derived LTR elements (within 20 kb) tend to show placenta-specific expression patterns, suggesting that THE1B elements are an abundant source of placenta-specific enhancers in primate genomes.The authors hypothesized that placental expression of human CRH expression. Given that placental CRH expression is unique to primates and the THE1B element is a primate-specific ERV, it would seem futile to model the regulation of this locus in mice or any other nonprimate animal. The authors sidestep this obstacle by creating mice with "humanized" placentas, with respect to the CRH locus. They generate transgenic mice harboring a stably integrated 180 kilobase pair-long bacterial artificial chromosome (BAC) encompassing the human CRH gene and upstream THE1B element. Mice typically only show CRH expression in the hypothalamus, but the transgenic mice showed robust and specific expression of human CRH in the hypothalamus and placenta, confirming their usefulness as a model for CRH regulation. Remarkably, mice expressing human CRH in the placenta were born on average 15 hours later that control mice, which coincides with the proposed role for CRH in regulating the timing of human birth.The authors next seek to experimentally investigate whether the THE1B element is indeed required for placental CRH in the placenta, the authors use clustered regularly interspaced short palindromic repeats (CRISPR) to delete the element from the human BAC sequences in the transgenic mice. In this second round of mutants, human CRH was still expressed in the hypothalamus, but its expression in the placenta was eliminated. Strikingly, deletion of the THE1B element fully rescued gestation length back to control levels. These experiments confirm that the THE1B element functions as a placenta-specific enhancer of CRH and suggest that the co-option of this retroviral element was an important step in the evolution of birth timing in primate pregnancy.To test whether the THE1B LTR element is required for expression of human distal-less homeobox 3 (DLX3) as a candidate factor, which is coexpressed with CRH, has a predicted binding site in the 5′ region of the THE1B insertion and is known to be required for placental development. The authors confirm that DLX3 physically interacts with the THE1B element using electrophoretic mobility shift assays and chromatin immunoprecipitation experiments in human placental tissue. Synthesizing their findings, the authors propose a model where the CRH gene is transcriptionally activated in the placenta through the binding of DLX3 to the THE1B-derived enhancer, which ultimately influences birth timing in pregnancy.Finally, the authors sought to determine the upstream transcription factors responsible for activating THE1B enhancer activity in the placenta. By analyzing transcriptomes generated from placentas of both human and rhesus macaque, they identify CRH expression were observed in transgenic mice, which do not normally express CRH in the placenta. However, without generating transgenic primates, the approach used in this study remains the most practical option for experimentally dissecting primate-specific gene regulation in the placenta.The study by Dunn-Fletcher and colleagues demonstrates that ERV LTRs can have biologically significant gene regulatory activities in the placenta and offers a new clue into the molecular basis of human birth timing. Their findings come with the caveat that the phenotypic effects of cytochrome P450 family 19 subfamily A member 1 (CYP19) gene and an enhancer for the human major histocompatibility complex, class I, G (HLA-G) gene [Several outstanding questions remain. First, how generally important are ERVs for placental gene expression? There is now strong evidence that ERV-derived proteins play an important role in placental development in many mammals, but whether ERV-derived regulatory elements play similarly significant roles remains unknown. In addition to the enhancer uncovered by this study, ERVs have also contributed a placenta-specific promoter for the human -G) gene ,28. AsidSecond, why is ERV transcriptional activity apparently elevated in placental cells? The placenta has long been observed to be a hotbed of ERV expression compared to the embryo, but the reasons for this remain poorly understood ,30. One Syncytin has been associated with human pregnancy disorders such as pre-eclampsia [CRH locus, due to noncoding genomic mutations or misregulation of upstream regulators such as DLX3, may be a contributing factor to premature or post-term birth. The co-option of LTRs as enhancer elements may also pose a conundrum for the cell and its intrinsic transposon silencing pathways, which must be tuned to permit the activity of beneficial ERVs while repressing potentially deleterious ones. Environmental or stochastic perturbations of these pathways during pregnancy may disrupt or activate genes with wide-ranging pathological effects.Finally, how do regulatory ERVs influence susceptibility to pregnancy-related complications? Misexpression of ERV-derived proteins such as clampsia . HoweverTo conclude, the study by Dunn-Fletcher and colleagues adds to a growing body of evidence supporting a remarkable evolutionary relationship between retroviruses and the placenta. A picture is now emerging in which important features of placentation are often reliant on proteins and regulatory sequences co-opted from ancient retroviruses . It is t


Incidence of cervical cancer among women of reproductive age still remains significantly high. In regard to prognostic features and risk factors, the standard treatment for most types of cervical cancer represents a combination of surgical treatment and radiation therapy, such as external beam radiation therapy and brachytherapy. Despite significant advances of long-term oncological outcomes, radiation-induced secondary malignancies among cervical cancer survivors are still an issue. Current case report describes an incredibly rare case of radiation-induced leiomyosarcoma of the rectum, which occurred 32 years after cervical cancer treatment.  A 62-year-old female had a past medical history of FIGO stage IIB cervical cancer (squamous cell carcinoma pT2bN0M0). In 1987, she underwent radical hysterectomy with bilateral iliac lymph node dissection, followed by adjuvant radiation therapy—70 Gy external beam pelvic irradiation followed by 30.5 Gy of brachytherapy. Thirty-two years later, she presented with signs of rectal bleeding. Regarding past medical history, radiologic, endoscopic, and pathologic data, the patient was initially diagnosed with a malignant nonepithelial lower rectal tumor of the unknown origin and staged as mrT3a mrN0 cM0. Total mesorectal excision with complete mesocolic excision and central vascular ligation (CME/CVL) carried by an open approach was carried out. In an attempt to identify the tissue of origin, an immunohistochemistry assay had been performed. Tumor cells showed a high rate of mitotic activity with a 45% rate of Ki-67 expression, positive reaction for desmin, and SMA in all samples. Negative reaction for CD117 and S100 was observed. As a conclusion, the immunophenotype was identified as a grade 3 leiomyosarcoma (ISD-code 8890/3).  We suggest that up to date, radical surgery with curative intent, as it was performed in our study, is the most evidence-based treatment option for patients with radiation-induced sarcomas of the rectum. Primary colorectal sarcomas are an extremely rare group of malignant mesenchymal tumors. They represent up to 0.1% of all primary diagnosed colorectal malignancies . RegardiA 62-year-old female had a past medical history of FIGO stage IIB cervical cancer (squamous cell carcinoma pT2bN0M0). In 1987, she underwent radical hysterectomy with bilateral iliac lymph node dissection, followed by adjuvant radiation therapy—70 Gy external beam pelvic irradiation followed by 30.5 Gy of brachytherapy. Thirty-two years later, she presented with signs of rectal bleeding.Digital rectal examination revealed a solid exophytic tumor on 5 cm above the anal verge. During rigid proctosigmoidoscopy, a 3 cm anterior wall rectal tumor with irregular margins and swollen mucosa was observed. Additional flexible colonoscopy showed no signs of synchronous colorectal neoplasms. Snare biopsy result showed an undifferentiated malignant nonepithelial tumor of unidentified histological origin. An attempt of immunohistochemistry assay was undertaken unsuccessfully due to an insufficient amount of tissue.Consecutively, a pelvic MRI with a 1.5 Tl Philips Intera machine had been performed for local staging. MERCURY protocol for high resolution imaging was applied.3b) was also identified. No radiological signs of either mesorectal fascia involvement or extramural vascular invasion (mrCRM− and mrEMVI−) had been observed . It was noticeable that the stump of the vagina and rectovaginal sept had prominent signs of fibrosis regarding a highly intensive MR signal on T2-weighted images. Malignant tumor features were also observed on a series of diffusion-weighted images  with complete mesocolic excision and central vascular ligation (CME/CVL) carried by an open approach was selected as proper extent of surgery. Standardized surgical technique in this case included complete left flexure mobilization, ligation of the inferior mesenteric artery at its origin near the aorta and inferior mesenteric vein just below the pancreatic tail with consecutive total mesorectal excision up to the level of the pelvic diaphragm see . AccuratOn postoperative day 5, the patient developed such clinical signs as nausea, vomiting, and loss of flatus and stool. Computed tomography of the chest, abdomen, and pelvis revealed multiple “levels” of gas and liquid throughout the small intestine with a breakdown of peroral contrast at the level of distal ileal loop, which was situated at the cavity of minor pelvis, adherent to the stumps of both the rectum and the vagina see . At thatBy taking in regard both clinical and radiologic findings, the patient was diagnosed with postoperative ileus and an immediate reoperation had been carried out. During abdominal examination, dilated intestinal loops with block of passage at the level of the pelvic diaphragm were identified see . An adheMacroscopically, the tumor was represented an exophytic homogenous lesion 45 × 25 mm with irregular margins and ulcerated surface covered with clots of fibrin. The tumor originated from the muscular layer of the rectum with concomitant mucosal invasion. Twelve regional lymph nodes were additionally examined. Initial histological appearance revealed a mixture of chaotic cellular and fibrotic vegetations. Among them, a subgroup of heterogeneous polykaryocytes with significant nuclear polymorphism  was identified see . Those aIn an attempt to identify the tissue of origin for a current tumor, an immunohistochemistry assay had been carried out. A panel of Ki-67, CD117, S100, smooth muscle actin (SMA), and desmin was used to conduct a differential diagnosis between leiomyosarcoma and gastrointestinal stromal tumor. Tumor cells showed a high rate of mitotic activity with a 45% rate of Ki-67 expression see . There wIn the current paper, we present the sixth case of radiation-induced leiomyosarcoma of the rectum which developed 32 years after completion of cervical cancer treatment. Throughout the last decades, significant success was achieved in the treatment of patients with pelvic malignancies, especially those who require either adjuvant or neoadjuvant radiation therapy. As a consequence of overall survival improvement, the problem of metachronous secondary malignancies among those patients who were exposed to radiation had arisen. According to the data of the National Cancer Institute of USA, over 40% of all patients diagnosed with primary malignancy will have to undergo radiation therapy . Most ofCervical cancer remains one of the leading cause of cancer-related morbidity and mortality among females . It is kIn cases of cervical cancer, the radiation field includes parametrium, sacrouterine ligaments, 3 cm of upper third of the vagina below the tumor, and presacral, obturator, external, and internal iliac lymph nodes. By taking in regard all the anatomical relations of the cervix, it becomes clear that the most critical radiation sites are represented by the vagina, the rectum, and the urinary bladder. In comparison, bony pelvis structures receive significantly lesser radiation dosages.A successful combination of curative surgery and highly precise modalities of radiation therapy in either adjuvant or neoadjuvant regimens had given a unique opportunity to improve cervical cancer patient's overall and disease-free survival rates like never before. Hence, by dropping out of a cancer recurrence group, those patients consequently enter another one—a risk group of developing a secondary malignancy. The first population-based study which highlighted a relevance between previous exposure to pelvic irradiation and a risk of developing a second malignancy was presented by Boice et al., in 1984 . The stuFirst studies about the influence of ionizing radiation on the development of malignancies were provided after a nuclear attack in Japan at 1945. Three main issues had been discovered then: tissues with higher proliferative index  are mostly affected; very small number of radiation-induced sarcomas and a ratio of 8%/1 Gray, which is a distribution of those who shall develop a malignancy among one hundred people exposed to an irradiation dosage of 1 Gy . MechaniAccording to the largest published series of Cha et al., only 125 (2.5%) out of 4884 primary soft tissue sarcomas satisfied Cahan's criteria and were considered as radiation induced . Vast maRemarkably, but up to date, there were only 5 published case reports of a radiation-induced leiomyosarcoma of the rectum in a patient after previous radiation therapy for cervical cancer see . On the Although radiation-induced leiomyosarcomas of the rectum appear to be an extremely rare type of secondary malignancies, there is an emerging data suggesting an intrinsically different sequence of molecular events responsible for their development and occurrence comparing to primary sarcomas . Gonin-LAmong all radiation-induced malignancies affecting long-term cervical cancer survivors, soft tissue sarcomas occur quite frequently. However, radiation-induced leiomyosarcoma of the rectum represents a remarkably rare case. Although primary colorectal sarcomas represent a very small subgroup of malignant mesenchymal tumors, the data regarding its proper treatment is lacking. On contrary to primary ones, radiation-induced sarcomas possess a number of unique molecular features which make them biologically different. We suggest that up to date, radical surgery with curative intent, as it was performed in our study, is the most evidence-based treatment option for patients with radiation-induced sarcomas of the rectum. Fibrotic changes in the pelvis as a consequence of previous external beam and brachytherapy may significantly jeopardise the completion of a TME procedure. That is why a two-step surgical strategy with delayed reconstruction should be taken in regard.


Peptide–nanoparticle conjugates (PNCs) have recently emerged as a versatile tool for biomedical applications. Synergism between the two promising classes of materials allows enhanced control over their biological behaviors, overcoming intrinsic limitations of the individual materials. Over the past decades, a myriad of PNCs has been developed for various applications, such as drug delivery, inhibition of pathogenic biomolecular interactions, molecular imaging, and liquid biopsy. This paper provides a comprehensive overview of existing technologies that have been recently developed in the broad field of PNCs, offering a guideline especially for investigators who are new to this field. Peptides have attracted a great deal of interest in biomedical fields as a novel material that can both exhibit protein functionalities and possess a high degree of modularity in molecular design. Current strategies for the discovery of artificial bioactive peptides can be broadly divided into two categories Fig. : (i) theOver the past decades, a large number of studies have demonstrated the utility of artificial bioactive peptides and some of these products have been successfully commercialized. Specifically, 28 noninsulin peptide drugs have been approved worldwide during the last two decades with several being highly competitive in the market . In addiMany researchers have found that the incorporation of peptides with non-biological materials  is a promising approach to addressing the intrinsic drawbacks of the peptides , 11. ParNanomaterials (tens to a few hundreds of nanometers in size) possess novel physico-chemical properties distinct from those of conventional bulk materials. Their ultra-small size and high surface-area-to-volume ratio are advantageous in the development of engineered materials that can uniquely interact with a variety of nano- and micro-sized biomaterials . The mosAnother important aspect that the PNCs can provide is multivalency. Most interactions in biological systems are based on non-covalent interactions such as hydrogen bonds, ionic bonds, van der Waals forces, π–π stacking forces, and hydrophobic interactions. Although the individual bindings are relatively weak, their co-operative action enables strong binding kinetics  based on the principle that the collective binding strength depends exponentially on to the number of individual binding pairs Fig. a 15–17]–1715–17]The presence of multiple binding sites plays a role in allowing the strong multivalent bindings and in increasing statistical opportunities for multiple monovalent binding events to occur. As depicted in Fig. The non-biological characteristics of NPs introduce novel properties and functions that are otherwise not obtained to their PNCs. For instance, NPs absorbing and emitting near infrared  light have been actively utilized in in vivo imaging due to the advantages of deep imaging depth and high spatial resolution . Some NPSelective delivery of pharmaceutical agents to target sites in the body remains a major challenge. Peptides have recently emerged as a powerful arsenal that may provide modular selectivity to drug delivery systems, warranting enhanced performance for the potential treatment of many serious health problems, such as cancer and brain diseases , 36. PepA simple approach to overcoming the problems of the current peptide-based delivery system is to combine them with NPs. Upon functionalization with peptides as targeting agents, NPs can be engineered to selectively deliver the drugs to the target tissue, in addition to their capability to encapsulate and protect therapeutic agents, increasing the plasma circulation time. As a result, researchers have conjugated different targeting peptides on different types of NPs to provide more efficient and adaptable drug delivery systems Table . One of Another interesting application for NPCs involves transdermal delivery for the treatment of melanoma. The main barrier preventing delivery for this application is the stratum corneum, the outermost layer of skin. Niu et al. designed a AuNP-based system that employed conjugated TAT peptides for the delivery of plasmid DNA (pDNA) . Their rl-lysines (DGL) NP conjugated with poly(ethylene glycol) (PEG) and a LIM Kinase 2 derived cell-penetrating peptide (LNP) for the delivery of pDNA across the BBB [Peptides have been also found to be useful for assisting NPs across other physiological barriers, including the blood brain barrier (BBB) that represents a major hurdle for effective delivery of pharmaceutical agents to the brain. The BBB acts as a shield surrounding blood vessels with access to the brain; its main purpose is to prevent non-essential substances from reaching the delicate system behind it . Researc the BBB . Their nThe PNC-based approaches have demonstrated a number of successful examples that have achieved efficient targeting to diseased cells and permeation across physiological barriers. However, there are many challenges that need to be overcome for ultimate translation of this approach, such as immunogenicity, long-term toxicity, and off-targeting potential. Upon addressing those concerns, it is foreseeable that the PNC approach will provide a powerful method for efficient drug delivery with high therapeutic index.Drugging the ‘undruggable’ targets is one of the key challenges in pharmacological studies . ApproxiPNCs provide a new insight to tackle these formidable challenges. For instance, the Lim group demonstrated inorganic NPs that serve as a scaffold for stabilizing peptide folding structures, which can eventually enhance both target affinity and selectivity . Figure The multivalent property of PNCs is a powerful tool for controlling polyvalent macromolecular associations that frequently occur in nature. Chaiken et al. reported that AuNP–peptide triazole conjugates inactivates HIV-1 by disrupting the interactions between host receptor proteins and trimeric envelope glycoprotein (Env) spikes of the virus , 57. As In some applications, PNC functionality can be significantly improved by precisely controlling the peptide valency. NPs that are covered with peptides at a higher density typically exhibit increased binding affinity ; howeverAnother advantage of PNCs is that they can utilize multiple therapeutic pathways by incorporating different types of molecules in a single nanoformulation system . RecentlAs described above, peptides have shown great potential to overcome their intrinsic limitations when conjugated onto NP surfaces. It has been reported that PNCs could outperform single peptides and even proteins, showing higher binding affinity, selectivity, and, in turn, therapeutic effect. This PNC approach has been also proven useful in other applications, such as molecular imaging and diagnostic/prognostic applications, including liquid biopsy, which will be discussed in the following sections.Molecular imaging provides visual information on biological processes at high resolution . It enabDespite their advantages, peptides often suffer from weak binding affinity, metabolic instability, and fast renal clearance due to their small size . These p2+ ions to generate strong NIR fluorescent signals [vβ3 integrin-specific RGD peptides to selectively image the tumor site. This tumor-specific imaging agent was highly photostable and showed a narrow emission spectrum, resulting in clear NIR fluorescent signals from the target tissue. Benzo-bis fluorophores have also been exploited for NIR imaging [NIR fluorescence imaging utilizes imaging agents with emission spectra in between 700 and 1100 nm . NIR lig signals , 82. Thi imaging . The fluco-glycolic acid) (PLGA)-AuNP [CT scans rely on multiple X-ray beams, generating cross-sectional images of bones, blood vessels, and soft tissues inside the body. AuNPs are one of the most commonly used imaging agents for CT scans, due to their high X-ray attenuation capability. Their biocompatibility, stability, and versatility enable AuNPs to be utilized in a wide range of applications . ConjugaGA)-AuNP . This NPGA)-AuNP , 87. Thi18F-labeled AuNPs to image the biodistribution of the targeted NPs [64Cu (64Cu-RGD-Au-tripods) to provide dual functionalities of integrin-specific targeting and PET imaging, respectively [125I and 76Br, respectively, demonstrating that the targeted nanoprobes exhibit enhanced cellular uptake, compared to non-targeted counterparts.PET is accepted as an excellent, non-destructive imaging tool for screening various diseases. Incorporation of target-specific peptides and positron emitters to NPs enables highly specific detection of abnormal tissues . CLPFFD eted NPs , 90. In ectively . The admectively . The binvβ3 integrin [MRI generates high-resolution three-dimensional images of organs and tissues using radio waves and magnetic fields . Magnetiintegrin . Their iintegrin , 96. Theintegrin . These c64Cu-labelled hollow gold nanospheres (64Cu-HAuNSs) were engineered to integrate CT and MRI capabilities into a single NP system [64Cu-HAuNSs to achieve selective targeting and increased cellular uptake of the NPs, resulting in highly selective dual imaging agent for both CT and MRI. Another common strategy involves the combination of fluorescence and MR imaging. Dendritic hybrid NPs, functionalized with activatable cell penetrating peptides (ACPPs) on their branches, were labeled with both Cy5 and Gd for fluorescence and MRI, respectively [Although PNCs have significantly improved the image quality of various modalities, more accurate and higher resolution imaging is necessary for early diagnosis and effective treatment. Multimodal contrast agents have been recently developed to help researchers and clinicians visualize two or more imaging modalities simultaneously. For example, P system . The RGDectively . ACPPs eThe examples described above clearly indicate the potential of PNCs as imaging contrast agents for a variety of modalities. Various peptides have been successfully employed for site-specific targeting in the field of biomedical imaging, which could be further improved using multivalent interaction of PNCs, providing high quality images of specific tissues and organs. Although their potential toxicity and biological instability need to be addressed for the successful clinical translation, PNC-based molecular imaging holds great promise to innovate current diagnostic and therapeutic platforms.Liquid biopsy is of high potential significance as a novel tool for diagnosis and prognosis of human diseases . It refeCirculating tumor cells (CTCs) , exosomeThis section summarizes several new technologies that use PNCs to detect liquid biopsy biomarkers with high sensitivity and specificity Table . Antibod4:1 detection specificity. Further clinical pilot studies revealed that the EGFR-specific PNCs detected up to 720 CTCs/mL from head and neck cancer patients’ samples.The Yang and Wang groups utilized peptides that recognize epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) for CTC isolation , 112. ThExosomes are endosomally derived extracellular vesicles (EVs) that play a major role in in intercellular signaling , 116. ItCirculating nucleic acids are another biomarker of interest, encompassing cfDNAs and miRNAs. The utility of circulating nucleic acids (NAs) has been investigated for several decades because the NA fragments that are released from the tumor may possess the entire genomic information of heterogeneous tumor cells . PeptideDespite lower binding affinity of free peptides, multivalent binding effect of the PNCs allows these short chain amino acid compounds to be utilized as capture agents for liquid biopsy with comparable capture efficiency to the devices using antibodies. However, the majority of PNC-based liquid biopsy platforms are still in the early stage of development; only a limited number of such devices have demonstrated clinical utilities. Further downstream analysis of the captured biomarkers, including molecular characterization and functional assays, would potentially enhance clinical applicability of the PNC-based liquid biopsy platforms.Molecularly poised between proteins and small molecular compounds, peptides can potentially exploit structural and functional advantages of the two major materials in pharmacological research. As summarized above, a number of peptides, combined with NPs, have shown that their promising potential in the area of drug delivery, inhibition of pathogenic biomolecular interactions, molecular imaging, and liquid biopsy. Despite the potential, clinical translation of PNCs still remains elusive due to the following reasons. First, the PNC behaviors in physiological conditions, such as bloodstream and intracellular space, have not been fully understood. Second, peptides are still vulnerable to enzymatic degradation even on nanomaterial surfaces , requiriUpon addressing those concerns, it is certain that the PNC systems would provide a novel class of materials that potentially fill the gap in current biomedical areas, such as drugging ‘undruggable’ targets, combating against multidrug resistant pathogens, isolating rare biomarkers from human body fluids, and utilizing as submicron-molecular imaging agents. Particularly along with the rapid advances in nanotechnology, the PNCs will likely become a new platform that can be used in mainstream therapeutic and diagnostic systems.


Hox transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. While the different Hox family members show clear functional specificity in vivo, they all show similar binding specificity in vitro and a satisfactory understanding of in vivo Hox target selectivity is still lacking.Drosophila Hox proteins. We find that Hox proteins show considerable binding selectivity in vivo even in the absence of canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility, being highest in less accessible chromatin. Individual Hox proteins exhibit different propensities to bind less accessible chromatin, and high binding selectivity is associated with high-affinity binding regions, leading to a model where Hox proteins derive binding selectivity through affinity-based competition with nucleosomes. Extradenticle/Homothorax cofactors generally facilitate Hox binding, promoting binding to regions in less accessible chromatin but with little effect on the overall selectivity of Hox targeting. These cofactors collaborate with Hox proteins in opening chromatin, in contrast to the pioneer factor, Glial cells missing, which facilitates Hox binding by independently generating accessible chromatin regions.Using transient transfection in Kc167 cells, we systematically analyze the binding of all eight These studies indicate that chromatin accessibility plays a key role in Hox selectivity. We propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of in vivo genomic targets for different Hox proteins.The online version of this article (10.1186/s13059-019-1721-4) contains supplementary material, which is available to authorized users. Although in vitro studies of transcription factor-DNA interactions have provided extensive insight into how transcription factors bind DNA –3, we haDrosophila Hox gene Ultrabithorax (Ubx) specifying development of the haltere balancer organ in the third thoracic segment which, in the absence of Ubx, develops as a wing . Eachiewed in ). A pote complex –12. In t complex –15. In s complex . However complex .Previously, we investigated the binding of selected Hox proteins in the context of chromatin through ChIP followed by high-throughput sequencing (ChIP-Seq) in Kc167 cells . We founDrosophila Hox proteins in the context of chromatin. We demonstrate that they each show distinct chromatin accessibility profiles and that high selectivity of Hox binding is associated with relatively inaccessible chromatin. In addition, we find that a major role of Exd/Hth cofactors is to promote Hox binding to relatively inaccessible chromatin. Overall, our studies indicate a key role for chromatin accessibility in determining the selective in vivo targeting of the different members of the Hox protein family.In this report, we present a more comprehensive analysis of the binding of all eight Drosophila Hox proteins using our previously established approach ),Overall, this analysis shows the relevance of both specific binding affinity, based on the quality and quantity of preferred motifs in binding regions, and chromatin accessibility for Hox protein target site selection. Binding to closed chromatin is associated with high TBA that may enable Hox proteins to effectively compete with nucleosomes. In addition, Hox binding occurs across a range of chromatin accessibility and here competition with chromatin may provide the potential for subtle differences in motif preference to generate different target sets for particular Hox proteins.We next examined the relationship between chromatin accessibility and the selectivity of Hox binding, as measured by the number of different Hox proteins binding to any particular region. We found a clear relationship, supporting a key role for chromatin accessibility in Hox selectivity. As shown in Fig. Hox selectivity is positively correlated with Hox TBA as seen in the general relationship between Hox selectivity and TBA for the individual Hox binding motifs Fig. d. The inOverall, the association of high Hox selectivity with relatively inaccessible chromatin and high-affinity binding regions indicates an interplay between affinity and chromatin accessibility in enabling Hox proteins to bind to different target sets. In a model of competition between nucleosomes and Hox proteins, binding to less accessible chromatin requires a higher affinity interaction between the Hox protein and the binding region. Accordingly, we find Hox binding regions in open chromatin show little discrimination, binding several or all Hox proteins. However, in less accessible chromatin, where competition with nucleosomes provides a basis for selective binding based on affinity, binding regions show more discrimination.In many situations, Hox proteins bind in association with the canonical Hox cofactors, Exd and Hth , 10. To The addition of Exd/Hth generally promotes Hox binding, and although the simple thresholded peak counts are not always increased . The cofactors increase the length of the enriched binding motifs and facilitate Hox protein binding to less accessible chromatin.Overall, we find that Exd/Hth has significant effects on in vivo Hox binding; for example, almost doubling the number of Dfd-bound regions . To study effects on chromatin accessibility using ATAC-Seq, we generated stable cell lines expressing representative Hox proteins, Dfd, Ubx, and Abd-B, since the transient transfections were not suitable for ATAC-Seq due to the dominance of plasmid sequences in the ATAC-Seq libraries. We compared the ATAC-Seq profiles of induced versus non-induced cell lines and found clear evidence that Hox proteins vary in their propensity to open chromatin. We see chromatin opening by Dfd and Abd-B Fig. a but notTo investigate the role of Exd/Hth, we generated stable cell lines expressing Dfd, Ubx, or Abd-B proteins together with Hth and found that the cofactors promote chromatin opening Fig. c. Examin−2, 13% of the total Dfd binding sites in the presence of Gcm), whereas for Ubx, Gcm has a larger effect, generating 4291 novel sites . The smaller effect of Gcm on Dfd binding may reflect the ability of Dfd to bind to less accessible regions on its own and we find that the presence of Gcm has little effect on the accessibility profile of Dfd-bound regions in comparison to the large effect for Ubx , which is believed to act as a pioneer factor . Kc167 cComparing the effects of Exd/Hth versus Gcm on Hox binding reveals two rather different routes to enhance Hox binding. In contrast to the Exd/Hth situation, the sites with significantly increased Hox binding in the presence of Gcm are associated with robust Gcm binding and chromatin opening by Gcm when expressed in the absence of Hox  on a scatter plot of accessibility (logATAC score) versus affinity (logTBA) Fig. b. The reEver since the initial analyses of DNA binding by Hox proteins in the 1990s , 28, 29,The propensity to compete with chromatin could depend on a variety of factors. The link between high TBA and high selectivity indicates that target selectivity could depend on differences in the affinity of interaction with binding sites. Hox proteins with relatively high affinity for their preferred binding sites, and/or with the ability to effectively use multiple binding sites to increase affinity, could compete with chromatin to establish binding at the relatively closed chromatin environments of the selective sites. Hox proteins with high numbers of unique binding regions, e.g., Pb, Dfd, and Abd-B, would have a high affinity for their preferred sites. On the other hand, Hox proteins, such as Antp and Ubx, would have a lower affinity and be unable to reach the affinity threshold for effective competition with chromatin and so would be restricted to binding less selective open chromatin regions. This could be termed a quantitative affinity model. Alternatively, selectivity could be based on more qualitative differences between Hox proteins, for example, they could differ in their ability to bind to nucleosomal DNA, or in their ability to interact with other DNA-binding proteins with whom they could collaborate to compete with chromatin , 30–32. We have investigated the influence of other DNA-binding proteins on Hox binding in chromatin in two different situations: provision of the canonical Hox cofactors Exd/Hth and provision of the pioneer factor Gcm. The Exd/Hth cofactors physically interact with Hox proteins through binding between Exd and the Hox YPWM motif and other interfaces –37. ProvAlthough Exd/Hth has a strong effect on the number and accessibility of Hox binding regions, we see the little general effect of Exd/Hth on the selectivity of Hox binding in vivo. However, particularly for Antp, Ubx, and Abd-B, the provision of Exd/Hth alters the Hox binding specificity as seen in the increased relative enrichment of the Abd-B motif (HoxB) versus the anterior Hox motifs (HoxA). This may occur through conformational constraints on Hox proteins in the Hox/Exd/Hth complex as with the phenomenon of latent specificity seen in vitro .The specific case of the interaction of Abd-B with Exd/Hth is interesting since Abd-B lacks the YPWM motif, although it may interact with Exd through other interfaces , and itsovo/shavenbaby locus in vivo where highly selective Hox binding is associated with low-affinity binding sites  were grouped by transcription factor and individual motifs plotted as dot plots with the median as colored bar or grouped into HoxA*  and Abd-B and plotted as boxplots using R. For total binding affinity (TBA) analysis [Motif enrichment analysis was performed using the R package PWMEnrich with the motifs from the MotifDb database . The motanalysis , the Hoxanalysis . For Hoxanalysis , matchPW.De-novo motif discovery was performed using HOMER  on the cConsensus matrixes for Fig. k-mers after each round.The top 15 prevalent 8-mer sequence patterns in Exd/Hth-enhanced binding regions were determined using Biostrings, masking identified SELEX raw data was downloaded from GSE65073  and reprq value 1e-2 was tiled into 20 bp windows and reads overlapping each window were counted using the csaw R package [The union of all regions bound by Hox or Hox+Hth at MACS  package . Reads wThe 10 bp adjusted ATAC-seq reads overlapping the 21,002 Hox group peaks (stable) regions were counted as above. These counts were then processed as for the cofactor-enhanced binding analysis. We defined significantly increased chromatin accessibility regions as edgeR fdr ≤ 0.01 and logFC ≥ 1.5 comparing induced versus non-induced samples  with chromatin accessibility (ATAC scores) or binding affinity (TBA), shown as scatterplots.Open chromatin regions in basal Kc cells  were tiled into 200 bp bins as follows: smaller regions were resized to 200 bp fixed on the center of each region and larger regions were split into 200 bp tiles. The tiles were then classified as bound or not bound if they overlapped a Hox bound region. Mean ATAC scores of basal Kc cells and TBA for HoxA or HoxB PWMs were calculated per tile. The log of these scores was then linearly binned into 40 bins, and a heatmap plotted. For the “All regions” plot, the heatmap colors show the location of the highest density of these tiles. The colors in the other plots represent the proportion of Hox bound within each bin. We then assessed the correlation Additional file 1:Figure S1. The association between Hox specificity and binding site affinity, binding region numbers and chromatin accessibility profiles; Figure S2. Hox, Hox+Hth, Hth and Exd binding in open and closed chromatin; Figure S3. Motif analysis showing individual motifs; Figure S4. De-novo motif analysis of Exd/Hth cofactor enhanced binding regions; Figure S5. Comparing the effects of Exd/Hth and Gcm: Chromatin accessibility in Hox + Exd/Hth compared to Hox + Gcm; Figure S6. Hox occupancy is more strongly associated with binding region chromatin accessibility than with binding affinity; Figure S7. The presence of Exd/Hth leads to both enhanced and reduced Abd-B binding; Figure S8. Comparison of Ubx and Dfd binding in presence of Gcm for regions in basal Kc167 closed chromatin; Table S1. ChIP-seq read overview; Table S2. ChIP-Seq binding region numbers; Table S3. Stable cell lines ATAC-seq read overview; Table S4. Cofactor-enhanced binding analysis of transient data in Hox group peak regions; Table S5. Increased chromatin accessibility analysis. (PDF 5942 kb)Additional file 2:Table S6. Hox group peak regions. (XLSX 3054 kb)