The ECHO trial randomised 7829 women to depot medroxyprogesterone acetate (DMPA-IM), the copper intrauterine device (IUD) and the levonorgestrel (LNG) implant (1:1:1) and found no clear difference in HIV incidence between these three groups. We have previously hypothesized that oligo-amenorrhoea induced by DMPA-IM may have a protective effect on HIV acquisition. The aim of this ancillary study was to assess the effects of DMPA-IM, the IUD and the LNG implant on menstrual symptoms and sexual behavior and to correlate these with HIV acquisition.At the Effective Care Research Unit (ECRU) in South Africa, of 615 women already randomised to DMPA-IM, the copper IUD and the LNG implant (1:1:1) 552 agreed to participate. Participants completed a 28-day symptom and behavior diary following their one-month ECHO trial visit and returning it at their 3-month follow-up visit. HIV acquisition data were retrieved from ECHO trial records.Of 552 women enrolled on the ancillary study, 390 (70.6%) completed their daily diary; 130, 133, and 127 received DMPA-IM, IUD, and LNG implant, respectively. Thirty-three (5.9%) of these women acquired HIV. Women on the progestin-only contraceptives were more likely to experience amenorrhoea, as expected, and were less likely to have intra-menstrual coitus than IUD users (p < 0.001 for DMPA-IM vs IUD and p = 0.002 for implant vs IUD). Overall coital frequency was highest and condom usage lowest among DMPA-IM users. Intra-menstrual coitus correlated positively, and duration of menstruation correlated negatively, with HIV acquisition, although these effects were not statistically significant .Findings support the hypothesis that oligo-amenorrhoea and the associated reduced intra-menstrual coitus may mitigate the potential for an increased biological risk of HIV acquisition with DMPA-IM but more evidence is needed.Study registration number PACTR201706001651380 There have been concerns that the depot-medroxyprogesterone acetate injection (DMPA-IM) may increase the risk of getting HIV infection. However, a large multicenter randomized study, the ECHO trial, recently compared HIV incidence among women randomized to DMPA-IM, the copper intrauterine device (IUD) and the levonorgestrel (LNG) implant and found little difference in HIV risk between these methods. DMPA-IM often causes no or scanty menstruation; we hypothesized that this may have a protective effect on getting HIV, by reducing exposure to HIV during menstrual bleeding.This ancillary study was done among ECHO trial participants at one of the ECHO study sites in South Africa. The aim was to assess the effects of the three different contraceptives on menstrual symptoms and sexual behavior and to correlate these with the risk of getting HIV. The study required women to complete a 28-day daily symptom and behavior diary after their one-month ECHO trial follow-up visit.We found that fewer women had sex during their periods with DMPA-IM and the LNG implant than the copper IUD, probably because no or scanty menstruation is more common with both DMPA-IM and the implant. Although effects were not statistically significant, having sex during periods tended to have a higher risk of getting HIV and longer periods indicated a lower risk of getting HIV.We concluded that sexual behavior related to menstruation may influence HIV acquisition and may partially explain why the ECHO trial found little difference in HIV incidence between the three contraceptives assessed despite observational evidence of higher biological risk with DMPA related to immune suppression. In Sub-Saharan Africa, injectable progestin contraception remains the most popular form of contraception, accounting for 45% of modern method use . HoweverThe role of sexual behavior in relation to contraception and HIV acquisition is not known. We have previously hypothesized that DMPA-induced oligo-amenorrhoea may have a protective effect on HIV acquisition by reducing sexual exposure during menstruation, thereby mitigating any potential biological increase in HIV risk , 13. UnpThus, the objective of this exploratory ancillary study to the ECHO trial was to compare the effects of DMPA-IM, the copper IUD and the LNG implant on intra-menstrual coitus and condom use, through the use of a daily symptom and behavior diary, and to correlate these with HIV incidence. We hypothesized that the progestin-only contraceptives would be associated with fewer events of coitus during menses, due to less menstrual bleeding, and more condom use, compared with the copper IUD; and that intra-menstrual coitus would be associated with an increased risk of HIV acquisition.This prospective study involved ECHO trial participants at the Effective Care Research Unit (ECRU) in East London, South Africa, one of the ECHO trial participating centres. The ECHO trial recruited women who were sexually active, HIV sero-negative between the ages of 16 and 35 years who were seeking effective contraception and compared the effect of three contraceptive methods  on HIV incidence and pregnancy rates . Partici®, Pfizer) given intramuscularly every 12 weeks, insertion of the subdermal LNG implant  or insertion of the Copper IUD . Of the 615 women that were recruited to the ECHO trial at ECRU, 552 agreed to participate in the daily diary ancillary study. Participants enrolled at the one-month follow-up visit were given a 28-day daily symptom and behavior diary to complete from the day of their enrolment visit and were asked to return it to research staff at the 3-month ECHO trial visit. To optimize compliance and the quality of self-reporting, research staff assisted participants to complete their ‘day 1’ entries. Participants were contacted daily by phone and reminded to complete their diaries. Parameters measured in the daily diary included sexual interest, sexual activity, condom use, mood and characteristics of menstruation ; pooled data for each study group was then expressed as mean values  with SD and compared using the t-test if the data are normally distributed. For non-parametric data, we used the Wilcoxon test to compare medians and interquartile ranges. We conducted a logistic regression analysis to evaluate the effect of the different variables on risk of HIV acquisition and present these data as Odds Ratios (OR) with their 95% confidence intervals (CI).A statistician (YB) from the South African Medical Research Council performed the statistical analysis using Stata Statistical Software V.15 . AnalysiOf the total sample of 615 women recruited at our centre to the main ECHO Trial, 552 agreed to participate in this daily diary study and 390 returned their daily diary with at least 27 completed days; 130, 133, and 127 received DMPA-IM, IUD, and LNG implant, respectively . There was a statistically significant difference between groups in the median number of days of menstruation, with women allocated to DMPA-IM and the LNG implant experiencing fewer bleeding days than those allocated to the copper IUD (p < 0.001) Table .Table 42Thirty participants recorded having no sexual intercourse during the 28-day period. These participants were equally distributed across the groups (p = 0.872). Women using DMPA-IM were more frequently engaged in sexual intercourse than women using the copper IUD (p = 0.015) or the LNG implant (p = 0.055), although the latter difference did not reach statistical significance. DMPA-IM and LNG implant users  were less likely to have intra-menstrual coitus than copper IUD users (64.7%) (p < 0.001 for DMPA-IM vs IUD and p = 0.002 for implant vs IUD).The proportion of women experiencing at least one day of severe menstrual pain was statistically significantly higher in the IUD group compared with DMPA-IM and the LNG implant, p = 0.035 and 0.018, respectively.The proportion of coital events covered by condom usage was significantly lower amongst participants using DMPA-IM . However, there were no significant differences between the groups in the proportion of women having at least one incident of unprotected coitus Table . There wThere was no difference in libido  or other parameters measured Table .Thirty-three women 8.5%) among our ancillary study cohort of 390 participants acquired HIV during the course of the ECHO trial. This incidence is similar to that of the larger cohort of 615 women who were recruited at ECRU for the ECHO trial. On logistic regression (n = 389), the number of occurrences of intra-menstrual coitus correlated positively with HIV acquisition , and duration of menstruation correlated negatively with HIV acquisition ; these associations did not reach statistical significance  was higher than that of the main ECHO trial (5%), highlighting the enormous risk of HIV infection among contraceptive users in our South African setting.As expected, oligo-amenorrhoea, a well-known side effect of progestin-only contraceptives, was much more common among DMPA-IM and LNG implant users than IUD users. As an apparent consequence of this outcome, intra-menstrual coitus was significantly more common among copper IUD users than users of the progestin-only methods. This was also a finding of the main ECHO trial, in which women were asked to recall at each follow-up visit whether they had had sexual intercourse during menses in the preceding 3 months . Among EIn our study, DMPA-IM users had the highest rates of amenorrhoea, the shortest duration of menstruation, and the lowest frequency of intra-menstrual coitus, probably because most women in this group experienced no menstruation during the study period. Conversely, copper IUD users had the lowest rates of amenorrhoea, the longest duration of menstruation and the highest frequency of intra-menstrual coitus. DMPA-IM users also had the highest coital frequency, which could be due to less days of menstrual bleeding and, therefore, more days available for coitus outside of menses. Higher rates of dysmenorrhoea may also have contributed to the lower coital frequency seem amongst copper IUD users in our study.The main ECHO trial found no difference in self-reported coital frequency between groups . HoweverDMPA-IM users in our study were less likely to use condoms than copper IUD and LNG implant users. This may be because condoms were more likely to be used when coitus occurred during menstruation. Copper IUD users had the highest rate of condom use with steady partners. Again, this may be because menstruating women were more likely to use condoms, and copper IUD users had more intra-menstrual coitus than the other groups.Our findings on reported condom use differ from those of the main ECHO trial, however, which found higher condom usage amongst the DMPA-IM users . This diWhilst no statistically significant associations with potential risk factors were found, the odds of HIV acquisition was increased with the occurrence of intra-menstrual coitus and reduced with longer duration of menstruation. Intra-menstrual coitus occurred in 49% of our study sample, indicating that this practice is common in our population. This is higher than found in another South African study that reported a lifetime incidence amongst 531 women of coitus during genital bleeding  of 26% . Women iOur finding of a tendency to a negative association between menstruation and HIV acquisition suggests that menstruation might be a protective factor for HIV acquisition, probably because women are less likely to engage in sexual intercourse during their menstrual periods and therefore have less sexual intercourse overall compared with women who do engage in intra-menstrual coitus. Women (and men) may intuitively feel more exposed to the risk of STI acquisition during menstruation and may prefer to avoid intra-menstrual coitus. In addition, decision-making about condom use during menses may be influenced by the desire to avert acquisition or transmission of STIs . ReducedThe United Kingdom’s National Health Service advises using a condom for sex during menstruation to prevent HIV and other STIs . HoweverThis ancillary daily diary study has several strengths: it was conducted alongside a rigorous RCT in which participants had been randomly assigned to contraceptive methods, therefore selection bias is unlikely to be responsible for behavioral differences. There were similar numbers of participants in each intervention group with similar baseline characteristics to the study center cohort; therefore, bias due to selective participation is unlikely. In addition, recall bias was minimized through the use of a daily symptom and behavior diary.However, it also has several limitations. We correlated HIV acquisition during the course of the ECHO trial with a daily diary commenced and completed during the second month after enrolment; a subsequent follow-up diary would have been preferable to confirm these findings. In addition, the lack of significance in the findings on intra-menstrual coitus may have been due to the small sample size.These findings support the hypothesis that oligo-amenorrhoea and the associated reduced intra-menstrual coitus may mitigate the potential for an increased biological risk of HIV acquisition with DMPA-IM. In addition, this study adds to the small body of evidence that suggests there may be an increased risk of HIV infection with intra-menstrual coitus. Education about the potential increased risk of acquiring HIV by engaging in coitus during menstruation should be considered, with condom use stressed as critical during this phase of the menstrual cycle in particular. More research into risk factors for HIV acquisition among contraceptive users is needed.


No installation or user registration is required. NAChRDBis accessible through any modern internet browser on desktops andmobile devices. By providing immediate and systematic access to practicalknowledge gained through decades of research, NAChRDB represents apowerful educational tool and helps guide discovery by revealing gapsin current knowledge and aiding the interpretation of results of molecularand structural biology experiments or computational studies.Nicotinic acetylcholinereceptors (nAChRs) comprise a large andancient family of allosteric ion channels mediating synaptic transmission.The vast knowledge about nAChRs has become difficult to navigate.NAChRDB is a web-accessible resource of curated residue-level functionalannotations of neuromuscular nAChRs. Interactive three-dimensional(3D) visualization and sequence alignment give further context tothis rich and growing collection of experimental observations andcomputational predictions. NAChRDB is freely available at  This function requires complex communicationbetween the agonist-binding pocket in the extracellular domain anda gating mechanism lying 60 Å away in a channel traversing theentire protein.6 This immense efforthas produced thousands of experimental observations and computationalpredictions for different receptor types, using inconsistent residuenumbering schemes and terminology. Furthermore, because nAChRs arelarge and difficult to crystalize, structural studies have focusedon individual parts of the molecule and neglected to account for theallosteric aspect, which is critical for nAChR function. In the absenceof comprehensive and unified structural annotation, it is almost impossibleto identify gaps in knowledge or areas of controversy and to promotefurther discoveries.8 NAChRDB addresses such limitationsby providing web-based, systematic access to curated residue-levelfunctional annotations of nAChRs, with interactive three-dimensional(3D) visualization and sequence alignment.Not surprisingly, nAChRs have remainedthe focus of intensive researchfor more than 50 years.9 Channel-lining residues were predicted using ChannelsDB.10 All predictions were added to NAChRDB as annotationrecords, with full reference to the source of information. Full detailsare available in the Supporting Information Methods.The modelsof complete nAChR structures were obtained from theProtein Data Bank, whereas the sequences of complete nAChR subunitswere obtained from UniProt. Functional annotations were collectedfrom a semisystematic literature review of Medline through PubMed1. All orFigure S1).NAChRDB currently contains approximately2000 unique annotation records describing the functional role of specificresidues, as inferred based on experimental observations or computationalpredictions reported for nAChRs of species from six genera. Thesedata come from 117 studies conducted between 1982 and 2019 at 92 institutionsfrom 25 countries. NAChRDB covers 41 methods and most annotationscome from electrophysiology, mutagenesis, or ligand-binding assays,as well as from computational methods such as molecular docking andrate-equilibrium free-energy relationship analysis .On the other hand, advanced searches can lead to important insightsthat can only be gained by examining individual findings in a largercontext. For example, while much of the knowledge about NAChRs hasbeen obtained via structural studies in ar nAChR 3A. Thus,otations 3B. Finalotations 3C. The f13 in the direction ofPDBe-KB,14 aiming to provide coherent accessto structural, pharmacological,and physiological data for nAChRs, without limitation to a certainorganism, tissue, type of receptor, or type of subunit. Most annotationscurrently stored in NAChRDB refer to neuromuscular nAChRs, but weare constantly updating the database and hope to increase coverageto all nAChRs and even nAChR-like proteins. As NAChRDB grows, we planto integrate with PDBe-KB through FunPDBe to promote visibility andstandardized access.NAChRDBgoes beyond A7DB,The relationship between publication andvalidation is central to the mission of NAChRDB, which is not to grantcredibility to the reports, but rather to create a venue for assessingthe credibility of reports in the proper context of the knowledgeaccumulated so far. In addition to annotations based on publishedstudies, NAChRDB currently also contains annotations based on thefindings of two computational investigations we performed in an effortto provide users with interesting opportunities for discussion andexploration. We hope that more researchers submit their findings toNAChRDB irrespective of the publication status because we firmly believethat the scientific community can only benefit from immediate accessto information, as long as the publication status of the findingsis clearly labeled. As such, every annotation record in NAChRDB containseither a DOI to the published source or a link to the source and theexplicit mention “Unpublished as of...”, to ensure transparency.NAChRDB is not simply a set of links to classifications, papers,or other databases, but a rich curated collection of relevant findingsthat describe structure–function relationships, uniquely suitedfor exploring the allosteric mechanisms underlying nAChR functionand pathology. The relevant information is given directly in the searchreport, which is not only informative but also helps spot contradictoryfindings and inconsistent reporting. Many residues highlighted bystate-of-the-art computational analyses have not been investigatedto date, mainly because experimental studies are expensive and thusfocus only on areas perceived to be of utmost importance. Furthermore,due to reporting bias, negative results are scarce. Thus, in additionto serving as an easy reference for structure–function information,we hope that NAChRDB will help promote the reporting of both positiveand negative results, so that the scientific community may form acomprehensive picture of nAChR functioning. Currently, NAChRDB representsa powerful tool both in classrooms and in labs focused on the studyof nAChRs. Ultimately, NAChRDB can also serve as a key starting pointfor unifying the state-of-art knowledge in the broad field of pentamericligand-gated ion channels.


Gaining information about the habitat environment and biological interactions is important for conserving gold-spotted pond frogs, which are faced with a threat of local population extinction in Korea due to artificial habitat changes. Based on stable isotope ratios, we estimated the ecological niche space (ENS) of gold-spotted pond frogs in an ecological wetland park and a rice paddy differing in habitat patch connectivity and analyzed the possibility of their ENS overlapping that of competitive and predatory frogs. Gold-spotted pond frogs showed a broader ENS in the ecological wetland park, wherein predation was relatively easy, than in the rice paddy. However, the ENS of the gold-spotted pond frogs was highly probably overlapped with that of other competing frog species that shared some of the food sources. Nevertheless, since the stable isotope analysis showed that gold-spotted pond frogs fed on more diverse prey than their competitors, it would remain relatively easy to procure alternative food sources, which are less affected by the competition in an environment with abundant food. Therefore, for stable settling of gold-spotted pond frogs into habitats and preserving their population, establishing habitat environments with highly diverse food sources is crucial, following consideration of their feeding behavior.Pelophylax chosenicus) is an endangered amphibian species in South Korea. In order to obtain ecological information regarding the gold-spotted pond frog’s habitat environment and biological interactions, we applied stable isotope analysis to quantify the ecological niche space (ENS) of frogs including black-spotted pond frogs (P. nigromaculatus) and bullfrogs (Lithobates catesbeianus) within the food web of two different habitats—an ecological wetland park and a rice paddy. The gold-spotted pond frog population exhibited a broader ENS in the ecological wetland park than in the rice paddy. According to the carbon stable isotope ratios, gold-spotted pond frogs mainly fed on insects, regardless of habitat type. However, the results comparing the range of both carbon and nitrogen stable isotopes showed that gold-spotted pond frogs living in the rice paddy showed limited feeding behavior, while those living in the ecological wetland park fed on various food sources located in more varied trophic positions. Although the ENS of the gold-spotted pond frog was generally less likely to be overlapped by that of other frog species, it was predicted to overlap with a high probability of 87.3% in the ecological wetland park. Nevertheless, gold-spotted pond frogs in the ecological wetland park were not significantly affected by the prey competition with competitive species by feeding on other prey for which other species’ preference was low. Since these results show that a habitats’ food diversity has an effect on securing the ENS of gold-spotted pond frogs and prey competition, we recommend that the establishment of a food environment that considers the feeding behavior of gold-spotted pond frogs is important for the sustainable preservation of gold-spotted pond frogs and their settlement in alternative habitats.The gold-spotted pond frog ( The niciversity ,3,4,5. Siversity ,7.Current understanding of niche width estimation is based on the heterogeneity of the feeding behavior of target organisms and ecological indices  . To cons13 C) provides insights into the food sources of a consumer, as the fixed value does not change significantly from photosynthesis through feeding, digestion, and assimilation, with a difference lower than 1‰ between prey and predator. In contrast, nitrogen (N) stable isotope ratios (δ15 N) are typically enriched in consumers relative to the concentrations in the diets, and the concentrations in predators can be up to 3.4‰ higher than those in prey [Stable isotopes have been used as alternative tools for analyzing food sources and overcoming the limitations of existing methods. These isotopes can accurately evaluate trophic structure based on the fractionation effect exhibited by each isotope during food assimilation into the tissues of consumers in the upper trophic levels ,8. The c in prey . A bi-pl in prey ,11. One 13 C–δ15 N stable isotope bi-plots are useful for identifying the isotopic niches of organisms within a food web and for the quantitative interpretation of community structure based on niche characteristics . However, stable isotope bi-plots have some limitations for assessing the impacts of individual species on trophic diversity and redundancy within communities. Layman et al. [13 C–δ15 N bi-plot and five metrics derived from it, δ15 Nrange (NR), δ13 C range, mean distance to centroid (CD), mean nearest neighbor distance (NND), and standard deviation of the NND (SDNND), reveals the responses of a population to environmental changes in the form of numerical values, which can be used not only to compare populations within a community, but also to analyze spatiotemporal changes in the overall community structure. However, community-wide metrics are estimates that do not consider uncertainties that may occur during sampling and population mean calculations, whereas TA, which forms the basis of community-wide metrics, is sensitive to sample size, presenting challenges when comparing populations (or communities) with different sample sizes. To address this problem, multivariate ellipse-based metrics, based on Bayesian inference techniques, that incorporate standard error in the estimates are used. Here, as standard ellipse area (SEA) can be derived by considering the sample size within a community , communities with different sample sizes can be directly compared [δcompared . Such stcompared ,12,13,14Pelophylax chosenicus)  have decreased in number in South Korea because of habitat destruction and the introduction of bullfrogs, an invasive species [All amphibians feed heavily on invertebrates; thus, their impact on this food source can be prodigious. In many habitats, amphibians are a major ‘conveyor belt’ that provides the transfer of invertebrate energy sources to predatory animals higher up the food chain. Amphibians have an important role in ecosystem energy flow and nutrient cycling in many ecosystems . Gold-sp species . The gol species . Gold-sp species ,18. In a species .Recently, programs to establish alternative habitats for conserving gold-spotted pond frog populations have been pursued ,21,22. AIn this study, we used stable isotope ratios to measure the niche widths of gold-spotted pond frogs and other frog species, with which they have competitive and predatory interactions, using frogs sampled from different habitats. The aim of the study was to investigate how different environments influence their feeding behavior and positions within the food web. Community-wide metrics were used to compare community characteristics, while niche overlap probability values were used to quantitatively assess how biological interactions with other frog species in different environments influenced gold-spotted pond frog trophic diversity. The results of this study provide basic ecological data on habitat environments and interspecific competition, which may facilitate the establishment and maintenance of gold-spotted pond frog populations in alternative habitats in conservation programs.Gold-spotted pond frogs are typically found in wetland environments. Therefore, when alternative habitats are created to protect their populations, the establishment of artificial habitats such as ecological parks is often considered . We seleNelumbo nucifera), common reed (Phragmites communis) and cattail (Typha angustifolia), and each pond is connected to a small water channel to facilitate free movement of the organisms along the waterways. Frogs in the rice paddy habitat were sampled mostly from rice paddies and waterways on the banks of Mankyeong River. Common reed (P. communis) was the dominant plant, and other hydrophytes, such as wormwood (Artemisia selengensis f. serratifolia), water chestnut (Trapa japonica), and silver grass (Miscanthus sacchariflorus) were found close by. The rice paddy habitat is vulnerable to habitat fragmentation because of concrete channels and human interference, including the use of farming equipment during cultivation seasons  to eliminate lipids to ensure that the C stable isotope ratio would accurately reflect food consumed recently [The captured frogs were ethically killed as painless as possible using MS-222 and then processed for analysis. Individuals sampled were transported to the Isotope Ecology and Environmental Science Lab, Hanyang University, under refrigerated conditions and subsequently freeze-stored (−23 °C) until stable isotope sample extraction. Appropriate amounts (1–2 g) of stable isotope samples were extracted from the muscles of each frog. Other tissues, such as bone, blood, and skin, were excluded to ensure accurate measurements. All samples were homogenized after freeze-drying (−85 °C) for at least 48 h, and then the samples divided into C and N stable isotope samples. The C stable isotope samples were treated with acid (HCl) to eliminate inorganic C . The samrecently ,31. No s13 C and δ15 N Appendix, C and N stable isotope ratios in the samples were analyzed using an elemental analyzer-isotope ratio mass spectrometer . Because the absolute quantities of C and N stable isotopes in the samples were very low, the isotope ratio was converted and expressed as a relative value compared to a reference standard. Vienna Pee-Dee Belemnite and air were used as the reference standards for C and N stable isotopes, respectively, and the abundances of the isotopes in the samples relative to in the reference standards were calculated as parts per thousand and expressed as δ13 C–δ15 N bi-plots that were constructed using the measured C and N stable isotope ratios. ENS values of frog populations between habitats were compared to determine their positions within the food webs, and differences in food sources and trophic levels of the populations were compared according to the habitat environments. ENS was calculated using the Stable Isotope Bayesian Ellipses function (SIBER) in R v3.5.1 based on the obtained stable isotope ratios [The ENS, or isotopic niche widths, of the frog populations, including the gold-spotted pond frogs in the two habitats, were analyzed based on δe ratios ,32. SIBEe ratios ,13. In aA with respect to species B was based on the overlapping area between the two species within the total niche of species B. Because interspecies-overlap has directionality, values measured in the reverse direction (niche overlap of species B with respect to species A) could also be derived. Niche overlap probability was calculated based on the degree of influence of the resource utilization capacities of the species, relative abundances within habitats, and C and N stable isotope ratios, enabling a comprehensive interpretation of the results [In order to evaluate the impacts of competitive and predatory interactions on the ENS of gold-spotted pond frog populations, the nicheROVER package in R was used to calculate the probability (%) of interspecies-niche overlap . Niche overlap of species The raw data of the C and N stable isotope ratios measured from the collected frogs specimen are summarized in 2 and 0.38‰2, respectively, indicating broader niches in the ecological wetland park habitat  and terrestrial insects  range from −28‰ to −23‰, whereas those of zooplankton and aquatic plants were reported to range from −34‰ to −29‰, and −30‰ to −26‰, respectively [Enallagma cyathigerum, Ephemerella dentata, Neocaridina denticulate, Neverita didyma, and Paracercion calamorum, Orders Odonata and Ephemeroptera) exhibited C stable isotope ratios ranging from −32‰ to −24‰ (unpublished data). When the C stable isotope ratio value ranges in the gold-spotted pond frog populations from the ecological park habitat were compared to those of the aquatic insects, the ranges were mostly similar, suggesting that these insects were the primary food sources of the gold-spotted pond frog populations in the wetland habitat.The stable isotope ratios of the gold-spotted pond frogs from the ecological wetland park habitat exhibited a wide distribution along the horizontal and vertical axes. The difference in N stable isotope ratios between trophic levels is generally 3.4‰ . ConsideIn contrast, gold-spotted pond frog populations from the rice paddy habitat exhibited narrow C and N stable isotope ratio ranges, indicating that their food sources were much more limited than that of the gold-spotted pond frogs from the wetland habitats, and that the individuals were largely feeding on similar food sources. The C stable isotope ratios ranged from −24‰ to −23‰, indicating very levels of low prey diversity. Given that there was little difference in the N stable isotope ratios among individuals these frogs may have fed on prey of similar trophic positions.Gold-spotted pond frogs are strongly omnivorous, feeding on either plant material or animals depending on feeding opportunities; therefore, their food sources vary depending on the environment . Food so2 and 0.42‰2, respectively, indicating a similar ENS regardless of habitat  and in the black-spotted pond frogs (0.42‰2) in the same habitat  of overlap with the ENS of the gold-spotted pond frogs in the ecological wetland park habitat, whereas such probability was very low (9.06%) in the rice paddy habitat. In contrast, gold-spotted pond frogs exhibited low probability of niche overlap based on the ENS of black-spotted pond frog in the ecological park habitat (4.6%) and rice paddy habitat (9.41%) A. AlthouGold-spotted pond frogs have a high preference for active food sources which have a low probability of being consumed by black-spotted pond frogs . TherefoThe above are some factors that may influence niche overlap. In addition, the influence of predators on other frog species and interspecific interactions should be considered, particularly in the case of the rice paddy habitats where bullfrog individuals were found. When ENS was compared between gold-spotted pond frogs and bullfrogs, a competitive relationship was inferred because the results did not reveal differences in trophic diversity B. The ovOur results highlight the need for environments with high trophic diversity when selecting or establishing alternative habitats for gold-spotted pond frogs. Here, trophic diversity is not limited to one ecosystem but applies to both aquatic and terrestrial ecosystems. Therefore, it is essential to create an environment in which preferred food sources can be accessed freely from the two ecosystems, with no ecosystem fragmentation. Further studies are required to investigate essential food sources for gold-spotted pond frogs that would increase their competitiveness in biological interactions when establishing alternative habitats. In addition, gold-spotted pond frogs exhibited a tendency to consume foods with relatively high N stable isotope ratio values as they grew, representing higher trophic diversity, whereas there was no correlation between growth and N stable isotope ratio values in the black-spotted pond frogs in a previous study . Such spWe used stable isotope ratios to analyze the ENS of gold-spotted pond frogs in two habitats to determine the influence of these habitats on feeding habits. The findings were applied to evaluate the influence of biological interactions based on comparisons with similar species. Measured C stable isotope ratios indicated that the sampled gold-spotted pond frog individuals fed on insects as their primary food source, regardless of the habitat, whereas N stable isotope ratios revealed slightly different distributions regarding trophic diversity depending on the habitat. Based on the habitat characteristics and stable isotope ratios, gold-spotted pond frogs in the ecological wetland park habitat with relatively high food abundance exhibited wider vertical ENS distribution, whereas those in the rice paddy habitat with somewhat limited food availability and individual mobility within the habitat exhibited narrow ENS with densely aggregated stable isotope ratio values.In conclusion, habitat environments with broad food source varieties were more favorable for securing ENS in gold-spotted pond frogs. The degree of niche overlap with competitive species was greater in the ecological park wetland habitat. Black-spotted pond frogs were sharing the ENS of gold-spotted pond frogs, and the degree of overlap with competitive species may have increased with the increasing diversity of food sources available to gold-spotted pond frogs within the ecological park habitat. Nevertheless, concurrently, the impact of niche overlap does not seem to have played a large role as gold-spotted pond frogs could also secure competition-free food sources. In contrast, in the case of the rice paddy habitat, where gold-spotted pond frogs and its competitors were estimated to exhibit limited feeding activity through narrow ENS, the competition among species may have been be intensified through continuous deterioration of the food environment, despite the probability of the calculated niche overlap being lower than that of the ecological wetland park habitat. In addition, bullfrogs in the rice paddy habitat exhibited greater competition than predation to the gold-spotted pond frogs; however, the overlap degree of not only the ENS but also of the C stable isotope ratio range indicated that food diversity was low, meaning that the bullfrogs did not strongly influence the feeding activities of the gold-spotted pond frog populations. Unfortunately, we used a relatively small number of samples for ENS analysis and could not directly investigate the available food sources in the habitat and the prey fed on by the frog species. Considering factors such as food preference analyses in addition to ecological characteristics (population density and distribution trends of frog species) within a habitat using quantitative data (stable isotope ratios in food sources) and their impacts on competition and feeding environments obtained from further study to supplement these limitations would reveal a more comprehensive picture of the biological and environmental factors present, and facilitate the establishment of more appropriate alternative habitats for conserving endangered species such as gold-spotted pond frogs.


Non-small cell lung cancer (NSCLC) was usually associated with poor prognosis and invalid therapeutical response to immunotherapy due to biological heterogeneity. It is urgent to screen reliable biomarkers, especially immunotherapy-associated biomarkers, that can predict outcomes of these patients.Gene expression profiles of 1026 NSCLC patients were collected from The Cancer Genome Atlas (TCGA) datasets with their corresponding clinical and somatic mutation data. Based on immune infiltration scores, molecular clustering classification was performed to identify immune subtypes in NSCLC. After the functional enrichment analysis of subtypes, hub genes were further screened using univariate Cox, Lasso, and multivariate Cox regression analysis, and the risk score was defined to construct the prognostic model. Other microarray data and corresponding clinical information of 603 NSCLC patients from the GEO datasets were applied to conduct random forest models for the prognosis of NSCLC with 100 runs of cross-validation. Finally, external datasets with immunotherapy and chemotherapy were further applied to explore the significance of risk-scores in clinical immunotherapy response for NSCLC patients.Compared with Subtype-B, the Subtype-A, associated with better outcomes, was characterized by significantly higher stromal and immune scores, T lymphocytes infiltration scores and up-regulation of immunotherapy markers. In addition, we found and validated an eleven -gene signatures for better application of distinguishing high- and low-risk NSCLC patients and predict patients’ prognosis and therapeutical response to immunotherapy. Furthermore, combined with other clinical characteristics based on multivariate Cox regression analysis, we successfully constructed and validated a nomogram to effectively predict the survival rate of NSCLC patients. External immunotherapy and chemotherapy cohorts validated the patients with higher risk-scores exhibited significant therapeutic response and clinical benefits.These results demonstrated the immunological and prognostic heterogeneity within NSCLC and provided a new clinical application in predicting the prognosis and benefits of immunotherapy for the disease.The online version contains supplementary material available at 10.1186/s12890-021-01765-3. As one of the most common tumors with high morbidity and mortality, lung cancer leads to a poor prognosis and increases critical social burden . Non-smaEGFR and ALK mutation testing to refine particular subtypes of NSCLC classification [PD-L1 associated mRNA signatures [In clinical practice, the TNM stage system was acknowledged as the most frequently used tool to predict the prognosis of NSCLC patients, which majorly depended on the inherent anatomical abnormity, including tumor size, lymph node situation and distant metastatic status . Howeverfication . Moreovegnatures , immune gnatures  and serugnatures , are emegnatures , 15. In gnatures . Hence, This study aimed to identify clustering immune subtypes of NSCLC and systematically assess the correlation between the characteristics of subtypes and prognosis, immunotherapy, and somatic mutation in NSCLC. Combining the prognostic gene signatures and classical clinical features, the risk model was established to improve predictive risk stratification and facilitate making a treatment decision for NSCLC patients. We are convinced that our findings would help to gain a further insight into the prognostic signatures of NSCLC and provide promising strategies for NSCLC immunotherapy.https://portal.gdc.cancer.gov/). The level 3 gene expression profile was integrated by the Illumina HiSeq 2000 RNA Sequencing platform and normalized as the FPKM form. The corresponding clinical data and somatic mutation data of these patients were also downloaded from TCGA for subsequent analysis, including age, gender, survival status and time, pathological stages, and TNM staging. Moreover, the microarray data and corresponding clinical information of 603 NSCLC patients were downloaded from the Gene Expression Omnibus (GEO) datasets (https://www.ncbi.nlm.nih.gov/geo/) as the external test datasets, including GSE37745, GSE31210 and GSE50081. The baseline clinicopathological signatures of these cohorts were summarized in Additional file The publicly available RNA-seq transcriptome data of 1026 NSCLC patients (including 522 LUAD and 504 LUSC) were collected from The Cancer Genome Atlas (TCGA) datasets (https://cibersort.stanford.edu/). Furthermore, based on the immune score, the “ConsensusClusterPlus” package [The ESTIMATE algorithm was applied to calculate the immune score for each patient using the R package “estimate”  and the  package  was used package  and “sur package . In addiTo functionally elucidate the biological characteristics of subtypes of LUAD and LUSC, the differential expression analysis was performed using the “limma” R package  and GeneTo further screen the prognosis-related genes of NSCLC, we conducted univariate Cox proportional-hazards regression analysis to preliminarily filter significant genes through using “coxph” function in “survival” R package. Subsequently, to remove the multicollinearity among these candidate genes, the LASSO regression was applied to screen independent prognosis-related genes with the optimal penalty parameter and the minimum 10-fold Cross-Validation . After fff value .To further clarify the characteristic of risk scores, we also performed multiple analyses based on high- and low- risk groups for 1001 NSCLC patients including Kaplan–Meier survival analysis, immune checkpoint analysis, clinical signatures comparison and immune infiltration analysis. Next, the multivariate Cox regression (stepwise model) was applied to construct the predictive model for NSCLC combined risk scores and other clinical features, including age, gender, immune subtypes, clinical stages, and TNM stages. Variables with p values < 0.05 were included into the Cox regression model and the nomogram was further constructed to predict the probability of one-, three- and five-year survival in NSCLC patients using “rms” package . To valiTo validate the prognostic value of risk scores in NSCLC patients, we re-performed Kaplan–Meier survival analysis and drew time-dependent receiver operating characteristic (ROC) curves using “timeROC” package  based onOther two independent datasets, GSE135222 and GSE126044, were used to estimate the curative response to immunotherapy, including 27 and 16 NSCLC patients receiving immunotherapy respectively. According to current clinical guidelines, some anti-tumor drugs have been recommended for NSCLC treatment including Cisplatin, Docetaxel, Etoposide, Gemcitabine, Paclitaxel, Pemetrexed, and Vinorelbine. To evaluate the therapeutic value of risk-scores in the chemotherapy treatment for NSCLC, we calculated the half maximal inhibitory concentration (IC50) value of above chemotherapeutic drugs based on Genomics of Drug Sensitivity in Cancer (GDSC) databases. Difference of IC50 value between high and low risk-score subgroups was compared using Wilcoxon test and the results were exhibited in box diagrams using the “ggpubr” package.https://www.r-project.org/). The continuous and categorical variables were presented as Mean ± Standard Deviation and number (percentages) respectively. Wilcox test was used to compare continuous variables and the Kaplan–Meier method was used to plot survival curves. The two-tailed p-value less than 0.05 was considered statistical significant.All relevant statistical analyses were performed in R software  and various mutation patterns were identified including missense mutation, nonsense mutation, nonstop mutation, translation start site, splice site and multi hit  in LUSC and 1283 DEGs (including 559 up-regulated and 724 down-regulated genes) in LUAD patients were identified respectively and 252 common DEGs were chosen for subsequent analysis  were identified in the risk signature based on their β coefficients   have been approved to treat NSCLC patients with genetic alterations in corresponding protein-encoding genes [As a malignant tumor with high mortality, the prognosis of NSCLC remains poor without an effective therapeutical response to immunotherapy due to tumor biological heterogeneity. In the past decade, identification of histological and molecular subtypes for NSCLC has resulted in dramatic improvements in disease outcomes . Particung genes . Howeverng genes . TherefoPD-L1, CTLA-4, LAG-3 and HAVCR2) have been demonstrated to play significant role in immune suppression in multiple tumors and several target inhibitors have also been widely applied to immunotherapy for cancers [In this study, we first proposed an immune molecular subtype based on clustering immune infiltration scores with distinct clinical and immunological signatures in LUAD and LUSC respectively. Interestingly, regardless of LUAD or LUSC, the characteristics of the two molecular subtypes manifested significant homogeneity. TME analysis revealed higher stromal and immune scores in SubA than that of SubB, indicating anti-tumor immune response was significantly activated in SubA of NSCLC . Moreove cancers . It was  cancers . In addiEGFR inhibitors might reverse of Warburg effect, one metabolic process of the excessive conversion from glucose to lactate in cancers, and re-activate oxidative phosphorylation of cancer cells for cancer therapy [To further explore the potential biological functional features of the subtypes in NSCLC, we also performed GO enrichment and GSEA analysis. Consistent with the immunological signatures of subtypes, functional enrichment analysis revealed that B cells associated biological processes including “B cell mediated immunity”, “Immunoglobulin mediated immune response” and “Cell activation involved in immune response” were more active in SubB groups while immunoregulation and amino-acid metabolism associated pathways such as “Positive regulation of leukocyte activation”, “Organic acid metabolic process” and “Peptide metabolic process” were significant enriched in the SubA cohorts. Markowitz’s study  has demo therapy . InteresFurthermore, to better clarify the prognostic value of DEGs for NSCLC, we successfully screened 11 prognostic risk signatures based on LASSO regression analysis and univariate/multivariate Cox regression analysis. High-expression of these risk signatures at protein levels was confirmed by immunohistochemistry from the HPA database. Notably, based on the expression of these genes, risk scores were further identified, which effectively stratified the NSCLC patients into high- and low-risk groups in TCGA and GEO dataset respectively. Survival analysis revealed that low-risk groups had longer overall survival than patients with high riskscores and ROC curves exhibited the certain predictive capacity of risk scores for the one/three/five years survival of NSCLC. Female had been recognized as the major cohorts for the never-smokers with NSCLC  and in tPD-1/PD-L1 inhibitors.In addition, high risk-scores were significantly negatively correlated with immune activation responses especially T cells activation through the immune infiltration analysis. The killing effect of CD8+ T cells especially cytotoxic T lymphocytes has been considered as the major effector cells in the anti-tumor process. CD8+ T cells could discriminate particular tumor-associated antigen and destroy cancer cells directly in various cancers, including oesophageal cancers , colorecTo further construct effective models for predicting the prognosis of NSCLC, we combined the risk prognostic signatures with other clinical characteristics based on multivariate Cox regression analysis. To better predict the one/three/five years survival of NSCLC for each individual, we successfully established the nomogram by incorporating age, gender, TNM stages and risk scores. Calibration curves exhibited that the nomogram had good prediction capacity in both three-year and five-year overall survival and its clinical practicability was also validated in DCA. Besides, data from three GEO dataset also confirmed that high-risk groups were associated with worse overall survival than low-risk groups with excellent AUC value. In the validation datasets, high-risk subgroups were also positive associated with female and high clinical stages and also discriminated NSCLC patients with poor outcomes. Furthermore, ROC curves showed that risk scores could be used to predict the prognosis for NSCLC combined with traditional clinical indices, with a high mean AUC value of 0.784. The results of our analysis using a ranking method with an RF model showed that risk-score was the most significant index for the prognosis of NSCLC, with greater mean decrease of Gini and Accuracy than other clinical indexes. These findings indicated that we might be able to evaluate and predict the prognosis of NSCLC through measuring the expression levels of the risk signatures to infer the risk scores.Furthermore, to validate the significance of risk-scores in the prediction of immunotherapy, the patients receiving anti-PDL1 immunotherapy were evaluated based on external datasets and we found the risk scores were significantly higher in patients responded to corresponding immunotherapy, suggesting target immunotherapy might be beneficial tool for the patients with high risk-scores. Besides immunotherapy, common chemotherapeutic drugs were also be demonstrated lower IC50 value in high risk-score cohorts, implying the high risk-score patients might be more efficacious against these chemotherapeutic drugs. Overall, these findings from external datasets validated the potential benefits in high risk-scores and indicated risk scores might play a vital role in predicting the curative responses to common chemotherapy and immune checkpoint therapy.However, there are still several limitations in our study. For one thing, although the risk prediction model for the prognosis of NSCLC was proposed and validated by TCGA and GEO datasets, the accuracy and clinical application of this model was still need more external congeneric researches, even clinical practices, to repeatedly confirm and improve. In addition, our study only found the association between the risk scores and poor prognosis in NSCLC while the detailed role of these risk genes in the pathogenesis of NSCLC remains to be further verified by in-depth in vivo and in vitro studies.In conclusion, our study firstly proposed the immune molecular subtypes based on clustering immune-cell infiltration scores with distinct clinical and immunological signatures in both LUAD and LUSC patients. Moreover, we identified and validated the immune risk prognostic model combined risk scores and clinical signatures, which can be used as an effective tool to predict the overall survival and immunotherapy efficacy of NSCLC. The various transcriptomic analysis helps us screen significant genetic signatures of NSCLC and provides a new clinical application in predicting prognosis and benefits of immunotherapy for NSCLC.Additional file 1: Fig. 1. The summary and description of the study workflow.Additional file 2: Fig. 2. Comparison of genomic alterations between SubA and SubB in the TCGA datasets. A, B Differential somatic mutation analysis of the two subgroups in LUSC patients. C, D Differential somatic mutation analysis between the two subtypes in LUAD patients.Additional file 3: Fig. 3.A, B Comparison of clinical phenotypes between two subtypes without significant statistical differences in LUAD and LUSC; C Comparison of other immune cells between high and low risk-score groups; D The expression of these risk genes remarkably increased in tumor patients using immunohistochemistry from HPA database. E Comparison of IC50 value between high and low risk-score groups for Paclitaxel and Pemetrexed.Additional file 4: Table 1. Clinical information of external validation datasets of NSCLC.


Arginine vasopressin (AVP) is expressed in both hypothalamic and extra-hypothalamic neurons. The expression and role of AVP exhibit remarkable divergence between these two neuronal populations. Polysynaptic pathways enable these neuronal groups to regulate each other. AVP neurons in the paraventricular nucleus of the hypothalamus increase the production of adrenal stress hormones by stimulating the hypothalamic–pituitary–adrenal axis. Outside the hypothalamus, the medial amygdala also contains robust amounts of AVP. Contrary to the hypothalamic counterpart, the expression of extra-hypothalamic medial amygdala AVP is sexually dimorphic, in that it is preferentially transcribed in males in response to the continual presence of testosterone. Male gonadal hormones typically generate a negative feedback on the neuroendocrine stress axis. Here, we investigated whether testosterone-responsive medial amygdala AVP neurons provide negative feedback to hypothalamic AVP, thereby providing a feedback loop to suppress stress endocrine response during periods of high testosterone secretion. Contrary to our expectation, we found that AVP overexpression within the posterodorsal medial amygdala increased the recruitment of hypothalamic AVP neurons during stress, without affecting the total number of AVP neurons or the number of recently activated neurons following stress. These observations suggest that the effects of testosterone on extra-hypothalamic AVP facilitate stress responsiveness through permissive influence on the recruitment of hypothalamic AVP neurons.The online version contains supplementary material available at 10.1186/s13041-021-00850-2. Continual interactions between the brain and endocrine system are required for appropriate response to environmental variability; a particularly robust example of this interaction is the reciprocity between stress and reproduction being a particularly robust example. Gonadal hormones typically suppress the stress endocrine response through negative feedback originating in the brain. Similarly, stress hormones from the adrenal glands suppress the reproductive hormones through both central and peripheral mechanisms. Thus, stress and reproduction can be conceptualized as two mutually exclusive neuroendocrine landscapes that allow an individual to exist in two separate behavioral states depending on the internal metabolic milieu and external environmental conditions.Testosterone, a male gonadal hormone, easily crosses the blood–brain barrier. It plays a role in regulating arginine vasopressin (AVP) transcription and hypothalamic–pituitary–adrenal (HPA) activity by acting through androgen receptors . VariousWith this background information, we aimed to investigate whether an experimental manipulation that increases AVP expression within the posterodorsal MeA causes the reciprocal attenuation of PVN-AVP recruitment during stress.Mice expressing Cre recombinase from AVP promoter (Cre+) and controls (Cre−) were infused with a viral vector in the posterodorsal MeA, delivering the AVP gene requiring recombination for its expression from a robust promotor . Similarly, AVP overexpression within the MeA did not cause a significant effect on PVN neurons showing recent activation in response to bobcat odor exposure . Thus, AVP overexpression within the MeA did not have a robust effect on the number of PVN AVP- or Fos-positive neurons. AVP overexpression within the MeA did not affect the number of PVN neurons expressing AVP . All animals with the Cre+  genotype exhibited more co-labeling than the maxima of Cre− littermates . To analyze whether the number of co-labeled neurons reflected the stochastic chance of encountering a Fos-positive and AVP-positive neuron, we calculated the expected frequency of co-labeling for each animal as the mathematical product of AVP and Fos frequencies. Expected frequency was then compared with the observed frequency of co-labeling by using paired t-test. Animals in the Cre+ group were found to exhibit co-labeling that was significantly higher than the stochastic expectation . In contrast, the expected and observed co-labeling frequencies did not differ significantly in the Cre− group . Thus, AVP overexpression within the MeA caused a greater-than-chance increase in the recruitment of PVN-AVP neurons after acute exposure to predator odor, without significantly affecting the total number of PVN neurons expressing AVP or those showing recent neuronal activity.We then compared the number of co-labeled PVN neurons, specifically quantifying those neurons that express AVP and have been active (Fos-positive) during recent exposure to bobcat odor. MeA-AVP overexpression robustly increased the number of co-labeled PVN-AVP neurons , and nuclear boundaries (DAPI) within the posterodorsal medial amygdala. Animals were surgically infused with viral vectors containing AVP within the posterodorsal medial amygdala to induce AVP overexpression. The expression of cargos was dependent on the availability of Cre recombinase in the genome of animals. Examples from Cre− and Cre+ are depicted with the scale bar of 10 μm. Fig. S2. Effects of experimental manipulation on paraventricular hypothalamus arginine vasopressin  neurons and those expressing the immediate-early gene . The number of positive neurons relative to the total number of neurons is depicted on the ordinate of panels a and b. Violin plots in these panels depict the median and inter-quartile range along with the raw values for all data points (n underneath the abscissa). Solid lines parallel to the abscissa in panel a and b represent the mean for the corresponding control.Additional file 2: Additional materials and methods.


Impaired cognitive reappraisal, associated with the social functioning and well-being of patients affected by mood or anxiety disorders, is characterized by distinct neural activation patterns across clinical populations. To date, studies dedicated to identifying common and distinct neural activation profiles need to be clarified. The aim of the present study was to investigate transdiagnostic differences and commonalities in brain activation patterns during reappraisal-mediated downregulation of emotions.Cognitive reappraisal of negative images was contrasted with maintaining emotions during a control viewing condition. Brain activation in 35 patients with major depressive disorder (MDD), 20 patients with post-traumatic stress disorder (PTSD), and 34 healthy controls (HC) during cognitive reappraisal was compared. Moreover, the neural circuitry of emotion regulation in these clinical populations was examined using seed-to-voxel and voxel-to-voxel functional connectivity analyses.Whole-brain fMRI analyses showed less right-lateralized activation of the inferior, middle, and superior frontal gyrus during cognitive reappraisal compared to viewing of negative images in MDD and PTSD patients compared to HCs. Right IFG activation was negatively correlated with the severity of anxiety and depressive symptomatology. In addition, increased seed-to-voxel connectivity of the right IFG as well as increased voxel-to-voxel connectivity was observed in PTSD patients compared to HCs and MDD patients.FMRI results therefore suggested a common deficit of depression and anxiety symptomatology reflected by reduced activation in right IFG during cognitive reappraisal as well as diagnosis specific effects in patients with PTSD based on seed-to-voxel and voxel-to-voxel connectivity showing an overactive and hyperconnected salience network. Findings highlight the role of transdiagnostic research to identify disorder specific brain patterns as well as patterns common across disorders.The online version contains supplementary material available at 10.1186/s12888-022-03780-y. The identification of neural mechanisms related to key clinical features in psychiatric disorders may help to tailor individualized brain-based training programs to treat cognitive deficits. For instance, the investigation of the neural underpinnings of emotion dysregulation and sustained negative affect as core features of anxiety and mood disorders may help identify neural targets for novel treatments such as real-time fMRI neurofeedback or transcranial direct current stimulation –3. CurreInvestigations of the neural bases of emotion regulation are critical to understanding the relation between a reduced capacity to use cognitive reappraisal and the severity of depressive symptoms . CognitiZilverstand and colleagues  extendedIn a similar line of investigation, Picó-Pérez and colleagues  studied In addition, the examination of the neural bases of volitional affect regulation in combat-related PTSD revealed that veterans with PTSD showed less recruitment of the dlPFC during cognitive regulation of affect, compared to veterans exposed to similar levels of combat stress without PTSD, suggesting altered neural activation during volitional self-regulation of negative affective states .Emotion dysregulation observed in MDD patients can also be considered a result of disruption of interconnected circuits. Dysfunctional connectivity of the affective salience, cognitive control, and default mode networks appears to underlie characteristic symptoms of depression, including depressed mood, anhedonia, self-rumination, and impaired concentration , 23. TheAltered emotion-related neurocircuitry has also been shown in individuals with PTSD. Sripada and colleagues  investigPTSD and depression are highly comorbid disorders, with approximately half of patients with current PTSD also showing co-occurring depression . FurtherThe aim of the present study was to investigate the fMRI co-activation patterns during reappraisal-mediated downregulation of emotion in MDD and PTSD patients compared to healthy individuals. Moreover, the neural circuitry of emotion regulation in these clinical populations using seed-to-voxel FC was explored. We expected to replicate common alterations in frontal regulatory control regions in both patient groups as well as observe disorder-specific alterations in limbic and parietal regions and also dorsal anterior cingulum. In addition, we sought to explore associations between anxiety and depression related symptom severity across patient groups as well as associations with specific types of emotion regulation strategies .Thirty-five patients with MDD (age 37.3 ± 13.5) and 20 patients with PTSD (age 43.4 ± 13.0) as well as 34 healthy individuals age 39.4 ± 12.1) without history of neurological or psychiatric disorders participated in this study. All participants were right-handed, Caucasian, had adequate knowledge of the German language, and normal or corrected to normal vision. Contraindications for MRI, pregnancy, as well as acute suicidality served as exclusion criteria for all participants. Diagnoses were based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) criteria  and were.4 ± 12.1The hospital anxiety and depression scale (HADS)  as well http://www.vislab.ucl.ac.uk/cogent.php). Participants were presented 19 pictures from the International Affective Picture System (IAPS) with negative valence  represented the activation of regions involved in the cognitive control of emotion, and the opposite contrast [reappraise < view] represented regions with lower activation for regulation compared to view during this task. Second-level results were evaluated with pFWE < .05 at voxel level for the individual groups and at p < .001 uncorrected voxel level and with p < .05 family-wise error (FWE) correction for the group difference (HC > Patients). Clusters were labelled using the automated anatomical labeling atlas 3 (AAL3)  to detect brain networks with increased activation during cognitive reappraisal compared to baseline (view), and [reappraise < view] to identify reduced activation patterns during cognitive reappraisal compared to baseline (view). Subsequently, the contrast images of each subject were entered into a second-level model. We performed a random-effect group analysis on the contrast images using one-sample 3 (AAL3) . To examp < .05) followed by post-hoc Tukey’s Honest Significant Difference (HSD) to correct for multiple comparisons. Post-hoc tests were done in STATISTICA Version 10 . At the selected threshold there were no significant differences between HCs and patients with PTSD  as well as between patients with MDD and PTSD. Furthermore, the three groups did not differ regarding lower activation during cognitive reappraisal compared to viewing.Furthermore, the same model was used to evaluate group differences between HCs and patients with MDD and PTSD. HCs had greater activation compared to patients (MDD + PTSD) in a cluster covering (according to AAL3) the right triangular 46.4%) and opercular (23.5%) parts of the IFG as well as the right precentral gyrus (17.4%) [(56 20 8), 6.4% and n = 4) of α = .0125. Significant negative correlations of HADS anxiety and depression scales with beta values  and HADS-Depression ) suggested that a higher clinical severity of anxiety or depression was related to less recruitment of the right IFG during cognitive reappraisal. A similar analysis revealed no significant association between mean beta values of the right lateral PFC ROI and the self-report of cognitive reappraisal  or suppression .To examine the relation of dysfunctional neural patterns during cognitive reappraisal and behavioral parameters, the cluster comprising the group difference (HC > Patients) was extracted and Pearson correlations between ROI mean beta values and ERQ as well as HADS results were computed. Significance was assessed based on a corrected threshold for the number of comparisons  showed that HCs had higher activation in the left MTC, PCG as well as dmPFC and SMA compared to patients with depression and higher activation in the right dmPFC and IFG compared to both patients with depression and PTSD.To evaluate task-related differences in FC between HCs and patients with MDD and PTSD, we conducted a seed-to-voxel connectivity analysis using the right IFG as a priori region of interest. To further investigate the functional organization of the brain during the cognitive reappraisal task without a priori assumptions, we conducted a voxel-to-voxel connectivity analysis.t-tests were computed for HCs as well as MDD and PTSD patients. HCs exhibited increased FC between the rIFG seed and the left cerebellum, left occipital fusiform gyrus, left lingual gyrus and precuneus  as well as the right pre/postcentral gyrus and the right superior parietal lobule . In addition, increased FC was found in an anterior network cluster comprised of the right superior frontal gyrus and the middle frontal gyrus . Patients with MDD demonstrated increased FC between the rIFG and the bilateral cerebellum as well as the left occipital fusiform gyrus . Reduced FC emerged between the rIFG and two clusters comprising the left middle frontal gyrus , the precuneus, left lateral occipital cortex, left middle frontal gyrus and posterior cingulate gyrus . PTSD patients showed increased FC between the rIFG seed and a antero-central network comprised of the bilateral paracingulate gyrus, the anterior cingulate gyrus, and bilateral medial and superior frontal gyrus .Using the right IFG (pars triangularis and opercularis) as seed, we investigated the effect of cognitive reappraisal compared to viewing of negative pictures on brain system interactions. For the between-conditions contrast (reappraise > view), separate Table As a data driven measure of functional network organization during the emotion regulation task, we investigated voxel-to-voxel connectivity. HCs showed a global reduction of voxel-to-voxel connectivity in a widespread network during cognitive reappraisal. The largest effect of voxel-to-voxel connectivity was present in a right-lateralized anterior network, which included the middle frontal gyrus, the paracingulate gyrus, the superior frontal gyrus, and the anterior cingulate gyrus. Patients with MDD showed reduced voxel-to-voxel connectivity in an anterior network including the supplementary motor cortex, the left paracingulate gyrus and bilateral superior frontal gyrus. Finally, the analysis showed no significant voxel-to-voxel connectivity patterns in PTSD patients during cognitive reappraisal see Fig. .Fig. 6Vot-tests , bilateral temporal, middle frontal, and superior frontal gyrus during cognitive reappraisal. Previous studies consistently report that cognitive reappraisal engages a prefronto-parietal network which exerts top-down control on limbic areas [Our whole brain fMRI analysis revealed significant activation of the bilateral inferior frontal gyrus  during downregulation of negative emotion . Wang anIn our study, we used strategies aimed at downregulating negative stimuli. Contrasting reappraisal versus view showed bilateral vlPFC activation in the control group. This is consistent with the pattern of right-lateralized activation for downregulation of negative emotions described by Ochsner and colleagues . The altIn addition to impairments across psychopathologies, Zilverstand and colleagues  reportedRegarding the neural correlates of emotion regulation deficits in PTSD, in addition to the reduced activation in the right IFG, the group of PTSD patients showed hypoactivation in the right dmPFC. Previous studies have focused on the structure, neurochemistry, and function of the amygdala, medial prefrontal cortex, and hippocampus in PTSD. Contrary to the hyperresponsiveness of the amygdala during emotion regulation, the medial prefrontal cortex appears to be volumetrically smaller and is hyporesponsive during symptomatic states and the performance of emotional cognitive tasks in PTSD . This fiFC analyses further investigated the importance of the group difference in the right IFG for cognitive reappraisal. In a seed-to-voxel fMRI connectivity analysis using the right IFG as seed region, PTSD patients exhibited altered FC compared to HCs and MDD patients during reappraisal. Compared to HCs, PTSD patients showed increased FC of the rIFG with the left frontal orbital cortex, the left temporal pole, the left inferior frontal gyrus (pars opercularis), and the left frontal operculum. Interestingly, our results indicated altered connectivity predominantly in the PTSD group, suggesting higher FC in a fronto-temporo-parietal network. Higher connectivity in this network may result from a compensatory mechanism, taking into account that regions are important for emotion regulation and more specifically cognitive control mechanisms . AlthougIn addition, PTSD patients showed greater FC than MDD patients in regions including the precuneus, the anterior and posterior cingulate gyrus and the bilateral paracingulate gyrus. This finding is consistent with reports of PTSD patients showing increased FC in the anterior cingulate gyrus as a core component of the salience network . This maTo investigate more general connectivity group differences, we employed a voxel-to-voxel connectivity analysis as a whole-brain voxel-based connectivity measure that represents how well connected any given voxel is to the rest of the gray matter voxels in the brain. Compared to the conventional seed-based connectivity analyses, voxel-to-voxel connectivity does not require a priori knowledge for the selection of the regions of interest . In the There are some limitations of the current study. First, the smaller sample size of PTSD compared to HCs and patients with MDD may have impeded the detection of significant differences between these groups. In addition, PTSD patients presented with relatively mild symptoms and whereas most individuals in the MDD group were treated on the ward, a high percentage of PTSD patients was treated in an outpatient clinic. In addition, different subtypes of emotion regulation in PTSD may complicate interpretation of the data. A previous review described a model that includes these two types of emotion dysregulation in PTSD . In thisPresented results underscored the importance of the lateral PFC for cognitive reappraisal across mood and anxiety disorders. Patients with MDD and PTSD showed reduced activation within the right IFG. Supporting a key role of this region in psychopathology, reduced right IFG activity predicted a greater severity of anxiety or depression in both patients with MDD and PTSD. However, seed-based FC of the right IFG and voxel-to-voxel connectivity patterns during cognitive reappraisal in PTSD patients were significantly different from those of HCs and MDD patients. In addition to hypoactivation of right IFG found in MDD and PTSD patients, these FC patterns suggested the underlying emotional processing of stimuli may show specific patterns for mood and anxiety disorders. Our findings underscore the importance of the lateral PFC for cognitive reappraisal and further suggest that the right IFG may be a suitable target for fMRI-based neurofeedback or other neuromodulatory interventions that may contribute to an improvement of symptoms.Additional file 1: Appendix 1. Regions demonstrating significant activations (reappraise > view) or deactivations (view > reappraise) in healthy controls, patients with MDD and patients with PTSD. Appendix 2. Between group differences in intrinsic connectivity as a measure of a voxel-analysis during cognitive reappraisal (reappraise > view).


A 40-year-old black male presented to ICU after intubation for airway protection due to rapid onset of neck weakness and swallowingdifficulty. His chest radiograph showed an unusual mediastinal opacity for which a computer tomography (CT) scan was done, confirminga mediastinal mass. Pyridostigmineis the preferred symptomatic treatment, and if patients do notadequately respond to this therapy, corticosteroids, azathioprine andthymectomy are first-line immunosuppressive treatments. There arefurther immunomodulatory drugs emerging, but they are limitedby the scarcity of controlled studies. Long-term drug treatment isessential for most patients and must be tailored to the particular formof MG.[3] Thymectomy is indicated as first-line therapy in all patientswith thymoma or suspected thymoma, regardless of the status of MG.Myasthenia gravis (MG) is a disorder of the neuromuscular junction(NMJ) which results in localised or generalised fatigable weaknessof skeletal muscles, commonly with ocular involvement.A 40-year-old black male, who was employed as a long-distance truckdriver, presented with a history of rapid onset of neck weakness,swallowing difficulty and ptosis. The treating physicians intubatedthe patient for airway protection, initiated antibiotic treatment fora suspicion of aspiration pneumonia and transferred him to anintensive care unit (ICU).On arrival in the ICU the patient had a temperature of 38.6 °Cwithout an obvious source of infection. His other vital signs werewithin normal limits. He was noted to have bilateral ptosis and a bulbarpalsy. Power in all limbs was normal. The rest of the examinationwas unremarkable. A chest radiograph During his admission to the ICU, a computedtomography (CT) scan of the patient’s chest was performed to explorea radiological abnormality, which revealed a well-circumscribed low-anterior mediastinal mass Anti-acetylcholine receptor (AChR) antibody testing was positive,and nerve conduction studies demonstrated a decremental patternin keeping with a diagnosis of myasthenia gravis. The patient wastreated with plasmapheresis, corticosteroids and neostigmine. He was successfully extubated after 7 days. Subsequent surgical excision of themediastinal mass confirmed a type B2 thymoma.[1] Whilecongenital MG results from gene mutations affecting the NMJcomponents, most patients who develop MG in adulthood haveautoantibodies specific to the postsynaptic AChR or functionallyrelated molecules.[4] These autoantibodies are thought to originatein hyperplastic germinal centres in the thymus where myoid cellsexpressing AChR are clustered.Myasthenia gravis (MG) is a disorder of the neuromuscular junction(NMJ) which results in localised or generalised fatigable weaknessof skeletal muscles, commonly with ocular involvement.[5] However, there is significantimmunological heterogeneity, with variation in antibody structure between individuals as well as between different muscles within asingle individual. Thymic abnormalities are present in the majorityof AChR antibody positive cases, with 60% - 70% of cases havingthymic hyperplasia and 10% - 12% having a thymoma.Autoantibodies are present in the serum in 80% to 90% of cases,most commonly anti-AChR antibodies.[6]Thymic epithelial cells produce unfolded AChR subunits that arebelieved to prime helper T cells to autoimmunity. These T cells thenattack the AChR on the myoid cells, creating infiltrating germinalcentres in the hyperplastic thymus and triggering complementactivation and deposition. The autoimmunisation is completed asthe antibodies in the germinal centres diversify to recognize intactmuscle AChR.The thymus contains a small number of myoid cells that arethe only known cells to express intact AChR outside of muscle.[7] The role of thymoma in autoimmunity is notclear, although it is thought that the histological subtype of thymomamay be important. The development of MG is associated with mixedthymomas, but not with thymomas of the cortical type. Certainantibodies are more commonly associated with the presence of athymoma.[2] In addition to AChR antibodies, some individuals havemuscle autoantibodies directed against titin or the ryanodine receptoras well as other intracellular muscle proteins. Among patients withMG, the presence of anti-titin antibodies is predictive of a thymicepithelial tumour .Anti-low-density lipoprotein receptor-related protein 4 (LRP4) andanti-muscle-specific kinase (MuSK) antibodies are not associated withthymomas.While only approximately 10% of patients with MG will have athymoma, it is known that up to a third of patients with a thymomawill develop MG.[8]Thymomas are also associated with an increased risk of developingother autoimmune diseases such as thyroiditis, rheumatoid arthritisand systemic lupus erythematosus.[7] Ifcomplete resection is not feasible, then a biopsy is required prior toneoadjuvant chemotherapy or radiotherapy, to improve likelihood ofsurgical resection. Patients with MG and a thymoma generally showsignificant improvement in disease after thymectomy. Moreover, thereis evidence for the benefit of thymectomy over drug therapy alonein patients with generalised MG and AChR antibodies, even in theabsence of a thymoma.[9] The thymectomy trial for non-thymomatousmyasthenia gravis patients receiving prednisone (MGTX), was amulticentre, assessor-blinded trial run between 2006 and 2012, whichenrolled 126 subjects with generalised AChR antibody-associatedMG.[7] It demonstrated significantly lower severity of weakness,lower prednisone and immunosuppressive agent requirements,reduced need for hospitalisation for MG exacerbations and a greaterproportion of subjects with minimal manifestations at 12 months inthe thymectomy group compared with the prednisone-alone group.Thymectomy is indicated as first-line therapy in all patients withthymoma or suspected thymoma, regardless of the status of MG.[10] Athymectomy should also be considered in children with MG.Evidence also favours thymectomy in early-onset disease ratherthan in late-onset disease, as the latter group of patients often havethymic atrophy and derive no benefit from the procedure.Certain populations should not undergo thymectomy basedon current evidence. Patients with MG and anti-MuSK or LRP4antibodies should not be offered thymectomy. In patients withpure ocular MG, there is insufficient evidence that surgery preventsgeneralisation of results in remission; however, it has been argued thatthymectomy should be considered in patients with ocular MG whendrug treatment has failed if the patients have AChR antibodies and arisk of generalised disease.[11] However, a differencewas noted in the clinical phenotype between the racial groups inthat black patients were more likely to develop treatment-resistantcomplete ophthalmoplegia and ptosis than white patients . Despite similar-sized cohorts, white patients weremore likely to develop generalised myasthenia poorly responsive totherapy (p=0.005) than black patients. There were no significant racialdifferences in the time between diagnosis to initiation of therapy, orthe performance and timing of thymectomy.A retrospective observational study published in 2007 demonstratedthat the annual incidence of anti-AChR-positive MG in the CapeTown metropole of South Africa is similar to that of developedcountries, without significant differences in the incidence ratesamong the three predominant racial groups.


Thirty-two percent of the reports had a misinterpretation of the masses as benign. Benign misinterpretations were more common in masses smaller than 5 cm (65% vs. 27%). Thirty percent of the reports suggested tissue biopsy and 6% recommended referral to a sarcoma center. MRI reports showed frequent deviations from ESSR guidelines, and protocol guidelines were not routinely met. Deviations from standard protocol and reporting guidelines could put patients at risk for inadequate therapy.Soft-tissue sarcomas (STS) are a rare subtype of soft-tissue mass and are frequently misinterpreted as benign lesions. Magnetic resonance imaging (MRI) is the primary recommended type of diagnostics. To assess the quality of primary radiology reports, we investigated whether recommended MRI report elements were included in compliance with European Society of Musculoskeletal Radiology (ESSR) guidelines. A total of 1107 patients were evaluated retrospectively, and 126 radiological reports on patients with malignant STS were assessed for ESSR quality criteria. One or more required sequences or planes were missing in 67% of the reports. In all 126 cases, the report recognized the mass as anomalous (100%). Sixty-eight percent of the reports mentioned signs of malignancy. The majority of reports ( Soft tissue sarcomas (STS) are a rare subtype of soft-tissue mass, representing less than 1% of all malignancies ,3,4. DueTo optimize the outcome for patients with soft-tissue sarcomas, treatment at specialized sarcoma centers is strongly recommended ,8,9,10. In cases of suspicious soft-tissue masses, magnetic resonance imaging (MRI) is the primary recommended type of examination. Therefore, subsequent radiologic reports play a crucial role in the detection of soft-tissue sarcomas and their following path of treatment . In any According to the European Society for Musculoskeletal Radiology (ESSR) guidelines, radiological reports on soft tumor masses should describe the exact anatomical location and use fluid-sensitive fat-saturated and T1-weighted sequences in at least two planes with the additional use of a contrast agent . FurtherThe aim of this study was to evaluate whether the aforementioned requirements stated in the current ESSR guidelines were included in the primary radiologic description of findings in patients that were subsequently treated for soft-tissue sarcoma in our specialized center.This study was approved by the ethics committee of the regional medical association (WF-095/20).A total of 1107 patients presenting to the traumatology and orthopedic surgery department of a tertiary sarcoma center for biopsy or excision surgery from 2012 to 2018 were evaluated retrospectively. Patients presenting to another surgical department of the sarcoma center were not included.n = 905). In some patient files, the initial MRI reports (n = 74) or final histopathological results (n = 2) could not be retrieved. Ultimately, 126 patients with complete initial MRI reports and final histopathological results were included in this study  were performed, which planes  were included, and was a contrast agent used?Was a conclusion drawn regarding the specific suspected diagnosis?Did the report mention a suspected malignancy or signs thereof? In cases of suspected “sarcoma” (see question 3), this question was answered with “yes”.Did the report include a recommendation for biopsy and histopathological analysis of the described tissue?Was a referral to a specialized sarcoma center recommended?The radiologic reports were analyzed for the following characteristics:Authors of MRI reports were listed, and their reports were counted to evaluate the number of contributed radiological reports from each radiologist or practice. Furthermore, the initial radiologically suspected diagnosis was compared to the final histopathological result to detect apparent mismatches. Mismatches were defined as differences in tumor dignity or in cases of a different suspected malignancy than the ultimate histopathological diagnosis. Vague suspected diagnoses  that were later specified into a subtype  by histological evaluation were not considered a mismatch.Statistical analysis was performed as a percentage-based evaluation, showing the proportions of cases in which the above-mentioned criteria were met.A total of 126 patients with a histopathologically confirmed soft-tissue sarcoma were included in our study. The study cohort consisted of 62 (49%) male and 64 (51%) female patients with a mean age of 63.1 (±17.5) years.n = 24; 19%), followed by myxofibrosarcomas , and myxoid liposarcomas ; no other radiologist was responsible for more than six reports. Most medical practices contributed one or two reports to our patient cohort.Radiological reports were obtained from 61 different radiological departments or medical practices. The largest share was from the in-house radiological department  phrased a specific suspected diagnosis. Thirty-two of these suspected diagnoses showed a gross mismatch with the final histopathological results, not counting reports in which the suspected diagnosis was broadly phrased and later specified through final histology .Regardless of their categorization of masses as benign or malignant, the vast majority of reports . Forty reports contained a gross misinterpretation of masses as benign (32%), although the masses were malignant soft-tissue sarcomas in pathological findings .n = 10) when in fact histology after biopsy or excision showed liposarcoma  or spindle-shaped sarcoma (n = 1)  than smaller masses . The most common mismatch was a suspected lipoma ( (n = 1) .Thirty-eight reports specifically recommended a biopsy of the described mass for further analysis (30%). Only 7 reports recommended a referral to a specialized sarcoma or oncology center for further diagnostics and/or treatment (6%).Our study showed that various quality criteria as outlined in current guidelines are frequently not included in initial MRI reports of soft-tissue sarcomas.Due to their rare incidence, many physicians, including primary-care physicians, orthopedic, plastic- or general surgeons, and radiologists that practice outside specialized sarcoma centers are only infrequently confronted with soft-tissue sarcomas . ClinicaOther, more common causes of soft-tissue masses include trauma, infection, benign lesions , or metastases. Accompanying symptoms such as fever, night sweat, or weight loss are only rarely observed . These uMRI facilitates not only detection, but also reliable differentiation between low-grade and high-grade soft-tissue sarcoma ,21. HoweProblems arise when soft-tissue masses are prematurely suspected to be benign. In our study, 40 reports (32%) did not articulate a suspected malignancy and 27 reports (21%) even described a specific benign tumor, whereas the final histological results proved malignancy. This inconsistency can have drastic consequences, as unplanned excisions of soft-tissue sarcoma often lead to incomplete resection and therefore hinder adequate disease control . The mosSurprisingly, “benign” misinterpretation is not only an occurrence with small-size tumors, as there were several cases of large masses (>5 × 5 cm) in which the primary assessment was “benign” but later corrected to “malignant sarcoma” through histopathological analysis.In the authors’ opinion, radiological reports should refrain from articulating a specific suspected diagnosis in cases of indeterminate soft-tissue masses. Instead, only signs of malignancy should be phrased, ideally in combination with the recommendation to obtain a tissue biopsy for further histopathological analysis. This process would be in line with the ESSR guidelines  and coulFurthermore, biopsies should be carried out in specialized sarcoma centers . This enThe large number of different radiologists who contributed to our patient cohort’s radiological reports suggests that the issue of non-adherence to ESSR guidelines is widespread and not due to the lack of awareness of a few radiologists.The broad variability in the conducted MRI sequences impedes the establishment of a common ground for diagnostics, interpretation, and treatment in cases of soft-tissue masses. Omission of certain sequences can lead to overlooked signs of malignancy. Additionally, incomplete plane reconstructions cause difficulty in the planning of surgical biopsy and especially ultimate excision, increasing the risk of subtotal excision or injury to surrounding structures.The relationship between patient outcomes with respect to survival, local recurrence, or complications after surgery and the effect of initial MRI protocols and report adherence versus non-adherence to ESSR guidelines remains unclear. An effect on patient outcome would suggest that magnetic resonance imaging should be repeated in accordance with ESSR guidelines prior to any further treatment.Only radiological reports based on conducted MRIs were evaluated in this study, because external MRI images themselves were not available in most cases due to lack of long-term file storage of external MRI images in our sarcoma center’s database. Image reevaluation by the authors and musculoskeletal radiology specialists would certainly provide further insights. However, for many primary care physicians or orthopedists, MRI reports, rather than the images themselves, are often the determining set of information used in deciding on further patient treatment or referral. This study reflects the data of the department of trauma and orthopedic surgery at a single sarcoma center. While the authors’ sarcoma center has a large catchment area in its region, evaluations of MRI reporting criteria might show different results in other countries or healthcare systems outside of the European continent with different existing guidelines. Furthermore, it is unknown whether some radiologists might have had other images  to compare the conducted MRI to. The presence of such additional information would most likely have affected their diagnostic accuracy.The present study identified substantial deviations of MRI protocols and reports from ESSR guidelines that may result in inadequate treatment of patients at risk. MRI reports in cases of soft-tissue masses should be based on a standardized protocol and written according to the guidelines, as such practices will be essential to ensuring high-quality reports and optimized care for patients in the future.