Background It has been observed that trauma patients have elevated plasma procoagulant activity that could be assigned to an elevated concentration of tissue factor (TF). However, in many instances there is a discrepancy between the levels of TF and the procoagulant activity observed. We hypothesized that factor XIa (FXIa) could be responsible for this additional activity and that the presence and levels of both proteins could correlate with trauma severity.Methods Citrate plasma from 98 trauma patients  were evaluated in clotting assays for the presence of FXIa and TF activity using respective inhibitory antibodies.Results When the three trauma patient groups were divided into two cohorts (Injury Severity Score [ISS] > 25 and ISS ≤ 25), higher frequencies and concentrations of both TF and FXIa were observed for all the more severe injury subgroups.Conclusions The majority of trauma patients have active FXIa in their plasma, with a significant fraction having active TF as well. Additionally, both TF and FXIa frequency and concentration directly relate to trauma severity. These data suggest the use of these two proteins as potential markers for the stratification of trauma patients. Worldwide, trauma is the leading cause of death and disability in individuals younger than 44 years old.One of the physiologic substrates of thrombin during the blood coagulation process is factor XI (FXI); however, FXIa can also be generated by FXIIa upon exposure to artificial surfacesIndeed, we observed in a previous study that the majority of trauma patients have both TF and FXIa present in plasma.Results from previous studies along with the exposure of TF to blood flow in trauma patients, as well as the inflammatory response to injury, suggest that both FXIa and active TF could be present in plasma from trauma patients and may correlate with injury severity. In the current study, our objective was to examine FXIa and active TF from groups of patients with blunt, penetrating, and thermal injuries with the hypothesis that, in response to injury, patients with greater injury will have greater FXIa and active TF in their blood.This study was reviewed and approved by the Institutional Review Board (IRB H-03–014) at the Brooke Army Medical Center (BAMC). All severely burned and injured trauma patients with or without inhalation injury admitted to the intensive care unit (ICU) were considered for enrollment. Because the study was considered to be less than minimal risk by IRB, no consents were required. Inclusion criteria were: patients ≥18 years of age admitted within 24 hours from injury and an anticipated stay of 72 hours or greater at the U.S. Army Institute of Surgical Research (USAISR) Burn Center or Trauma ICU at BAMC. Patients and their families were given an information sheet concerning this less than minimal risk study prior to their enrollment. The following patients were excluded from the study: injury greater than 24 hours, prisoners, patients admitted to the wards, showing clinical signs of active hemorrhage, died within 72 hours after injury, patients undergoing therapeutic anticoagulation including Coumadin or antiplatelet agents, and those with known underlying coagulopathy. In addition, eight healthy volunteers were recruited and used as normal controls.Baseline blood samples were collected into citrate tubes  within 24 hours of admission (define as “Day 0”), followed by blood draws at 1, 2, 3, 5, and 7 days after admission. All samples were taken from an arterial or venous line inserted for standard clinical care. At the time of sample collection, no heparin was used in either arterial or venous lines due to the risk of heparin-induced thrombocytopenia. All the processing of blood samples was performed by trained research coordinators, at the clinical research laboratory of USAISR, which is located in the same facility as the Brooke Army Medical Center. Citrate blood-containing tubes were centrifuged immediately at 3,000 g for 15 minutes at room temperature and citrate plasma was frozen and stored at −70°C until assayed.2was added to a final 15 mmol/L concentration and the plasma incubated for 1 minute; clotting was initiated by the addition of 2 μmol/L phospholipid vesicles (PCPS) composed of 25% dioleoyl-sn-glycero-3-phospho-L-serine and 75% of 1,2-dioleoyl-sn-glycero-3-phosphocholine  and prepared as described previously.2addition. αFXI-2 is specific for FXI/XIa and inhibits FIX activation by FXIa.1–242 in pooled 10-donor normal plasma.These assays were done as previously described.p-values less than 0.05 were considered statistically significant.Categorical demographics and presence of TF or FXI at baseline were reported as frequency (%) and compared across trauma types using the chi-square test. Continuous demographic variables were reported as median (interquartile range [IQR]), and raw FXIa levels were reported as 90th percentile (75th–100th). All comparisons of continuous variables across trauma types were made using the Wilcoxon rank-sum test. Alln = 212) had an average PT value of 10.7 seconds , an average aPTT value of 29.3 seconds , and an average fibrinogen value of 571.9 mg/dL . Penetrating patient samples (n = 61) had an average PT value of 10.8 seconds , an average aPTT value of 33.5 seconds , and an average fibrinogen value of 560.4 mg/dL . Thermal patient samples (n = 174) had an average PT value of 12.3 seconds , an average aPTT value of 36.3 seconds , and an average fibrinogen value of 532.1 mg/dL . The eight healthy donors enrolled had an average PT value of 10.4 seconds , an average aPTT value of 28.3 seconds , and an average fibrinogen value of 307.7 mg/dL . Eleven patients with thermal (34%), 5 with blunt (11%), and 1 with penetrating (6%) trauma  died during hospitalization. Seven of 11 thermal patients with a lethal outcome had third degree burn for >10% of their body surface and 5 of 11 had inhalation injuries. Four of five blunt trauma patients who died had head–neck injuries . The death of the single patient with a penetrating trauma was caused by a chest–thorax injury (AIS = 5). For patients with a lethal outcome, the mean survival time after admission was 23 ± 38 days .From April 1, 2004, through May 31, 2005, a total of 479 burned and 1,366 nonburn trauma patients presented to the emergency department, and were screened for enrollment into study. Of these, 579 (212 burned and 367 nonburn trauma) patients were admitted to the ICU and blood samples obtained at specified time points. Three patients were subsequently excluded because they died within 48 hours after injury. Of these patients, 98 trauma patients had their plasma analyzed for TF and FXI activities. They were chosen based on mechanism of injury, divided into three groups based on the type of injury . Groups were comprised of 34 thermal patients , 47 blunt injury patients, and 17 penetrating injury patients . Overalp-values between 0.05 and 0.01 and hospital days and injury to head reported as significant withp-values < 0.01.Statistical analysis was performed across trauma groups for all demographic parameters presented inOverall, 468 samples from 98 patients were processed for TF activity , with 3Blunt injury patient samples comprised 48% of baseline samples and 47% of the total number of samples analyzed. From the blunt injury baseline samples, 26% had active TF resulting in a mean of 0.26 ± 0.44 pM . TF freThe smallest number of patients and corresponding samples was in the group of penetrating injury patients, which made up 17% of total baseline samples and 14% of samples overall. At baseline, 29% of penetrating injury samples had active TF resulting in a mean of 0.43 ± 0.27 pM. Similar to blunt injury patients, the frequency of TF in penetrating injury samples decreased with time from 29% in baseline samples to 21% overall (59% of penetrating patients) resulting in a mean of 0.27 ± 0.57 pM.Thermal injury samples comprised a similar number of baseline and overall samples as blunt injury samples . Thirty-five percent of baseline samples taken from thermal injury patients had active TF resulting in a mean concentration of 0.35 ± 0.49 pM, while, similarly to the other two groups, only 15% of samples overall  had active TF resulting in a mean concentration of 0.15 ± 0.36 pM. Of the nine thermal patients presenting with inhalation injury, five patients had TF present at baseline resulting in a mean concentration of 0.56 ± 0.53 pM, while overall the mean concentration was 0.2 ± 0.44 pM.p-value of 0.64 was obtained, indicating no statistical significance. Statistical analysis was not performed for the frequency of TF across all samples as patients had varying numbers of observations, nor was it performed for TF concentrations given that the majority of samples with TF present were equal to 1 pM and it could not have been treated as a continuous variable.When the frequency of TF at baseline was compared across blunt, penetrating, and thermal injury trauma groups , aOf the 98 baseline samples taken upon Day 0, 46% had detectable FXIa with a mean concentration of 19.6 ± 32.2 pM, whereas only 23% of the total 468 samples collected had detectable levels of FXIa  with a mean concentration of 9.0 ± 22.5 pM . None oFor blunt injury patient baseline samples, 53% had detectable FXIa resulting in a mean concentration of 20.3 ± 27.3 pM. Similar to TF, the frequency of FXIa decreased with time, and for all samples analyzed from blunt trauma patients, 219 of total 468 samples (29%) had detectable FXIa (66% of blunt patients) resulting in a mean concentration of 10.3 ± 23.0 pM.The smallest patient group was those who had penetrating injuries; 24% of ICU Day 0 samples contained detectable FXIa resulting in a mean of 19.2 ± 48.1 pM, whereas of all penetrating trauma samples  19% had detectable levels of FXIa (47% of penetrating patients), resulting in a mean of 10.6 ± 30.9 pM.For patients with thermal trauma, 47% of Day 0 samples contained detectable FXIa, resulting in a mean of 18.8 ± 29.9 pM. For all thermal trauma samples , 20% had detectable FXIa  resulting in a mean of 6.8 ± 18.1 pM. Of the nine thermal patients presenting with inhalation injury, six patients had active FXIa present at baseline, resulting in a mean concentration of 29.7 ± 28.0 pM, while overall the mean concentration was 11.4 ± 21.6 pM.p-value of 0.11 was obtained, indicating no statistical significance. Similarly, when the 90th percentile of FXIa concentrations at baseline was compared across blunt, penetrating, and thermal injury trauma groups , ap-value of 0.21 was obtained, indicating no statistical significance. Statistical analysis was not performed for the frequency of FXIa across all samples as patients had varying numbers of observations, nor was it performed for all FXIa concentrations given that so many samples had a value of 0 pM.When the frequency of FXIa at baseline was compared across blunt, penetrating, and thermal injury trauma groups , an = 47), penetrating (n = 17), and thermal (n = 34), was split into two subgroups based on those with ISS > 25 and those with ISS ≤ 25. For these subgroups, only baseline samples were analyzed for the frequencies of TF and FXIa and mean values . Similarly, a 1.8-fold higher prevalence of FXIa was observed for the blunt patient subgroup ISS > 25 (36.8 vs. 64.3%), with frequencies higher than those observed for TF. In regard to TF concentrations, the same pattern was observed with a twofold higher concentration in the ISS > 25 blunt patient subgroup (0.16 ± 0.4 vs. 0.32 ± 0.5 pM). Additionally, the same pattern was observed for FXIa in which concentrations were 1.8-fold higher in the blunt patient subgroup ISS > 25 (13.5 ± 22.8 vs. 24.9 ± 29.5 pM) .In contrast to blunt patients, the least pronounced difference in TF frequency in subgroups ISS ≤ 25 and ISS > 25 was observed for penetrating patients . FXIa frequency in penetrating patients was at a lower frequency than that in blunt patients, although the 1.8-fold difference in subgroups ISS ≤ 25 and ISS > 25 remained consistent . For TF concentrations in the penetrating patient subgroups ISS ≤ 25 and ISS > 25, there was only a 1.3-fold increase as opposed to the twofold increase observed for blunt patients, although slightly higher mean values were obtained . A slightly lower increase (1.6-fold) was also observed for FXIa concentrations in ISS ≤ 25 and ISS > 25 subgroups of penetrating patients versus blunt patients, although with similar concentrations .Thermal patients had the highest frequencies of both TF and FXIa in either subgroup. For TF in subgroups ISS ≤ 25 and ISS > 25, there was a 1.7-fold increase in frequencies  and also a 1.7-fold increase in mean values . For FXIa in subgroups ISS ≤ 25 and ISS > 25, a 1.9-fold increase in frequencies was observed  along with a 1.5-fold increase in mean values .This study evaluates the prevalence and levels of TF and FXIa within 24 hours of injury and during the course of the hospital stay for groups of blunt, penetrating, and thermal injury trauma patients. Although thermal injury patients had the highest Day 0 frequency of TF, penetrating injury patients maintained the highest overall frequency along with the highest TF levels in both Day 0 samples and in all samples analyzed.n = 17) with less than half having ISS > 25, and the fact that they had the lowest mean ISS score of all. The lack of a distinguishable and significant pattern of TF prevalence and/or concentration over time is consistent with a previous studyWhen all three trauma groups were subdivided into ISS ≤ and ISS > 25, there was a twofold higher TF frequency and concentration in the higher ISS subgroup for both blunt and thermal trauma patients. In contrast, frequency and concentration were only 1.2- and 1.3-fold higher, respectively, in the penetrating trauma subgroup of ISS > 25 as compared with ISS ≤ 25. It is perhaps expected that this group of penetrating injury patients would have less of a correlation between TF and ISS due to sample size, as they comprised the smallest of all groups  samples and also during the entire hospital stay was in the group of blunt injury patients, whereas the lowest frequencies in both sample sets were observed in penetrating patients. In terms of mean FXIa levels, however, there was no pronounced difference in all three groups. The elevated frequency of FXIa in blunt patients compared with thermal and, even more pronounced, to penetrating patients may be explained by injury mechanism and the subsequent elevation of apoptosis. The most common causes of blunt trauma injuries are due to motor vehicle collisions, falls, industrial/recreational accidents, etc., in which a large number of patients suffer from traumatic brain injury and/or pulmonary injury—both of which are shown to lead to increased apoptosis and the subsequent release of histones and polyphosphates into the extracellular space.When each group of trauma patients was divided into the subgroups ISS > and ISS ≤ 25, there was a similar higher prevalence of FXIa in high ISS subgroups across all three injury groups. That is in contrast to that observed for TF, in which penetrating patients responded less to severe versus nonsevere injury compared with blunt and thermal patients. Similar to frequency, each group had higher levels of FXIa for subgroups of ISS > 25 although no pronounced difference was observed in the increase across blunt, penetrating, and thermal patient subgroups.There are several limitations in this study. (1) Analysis was focused on TF and FXIa and we did not simultaneously assess other potential biomarkers that could provide further insight to trauma-induced coagulopathy (TIC). However, we have already published our observations of derangements in whole blood (thromboelastography [TEG]) and plasma .In conclusion, this study demonstrates that FXIa and active TF are quantifiable in the plasma derived from blunt, penetrating, and thermal injury trauma patients; their concentrations relate to injury severity. FXIa and active TF could therefore serve as potential new biomarkers of trauma severity and detection of these proteins may ultimately assist in the improved treatment for trauma patients. Future studies to characterize the profiles FXIa and active TF over time, taking into account shock, hypotension, and transfusions, would enhance our understanding of their role in TIC after injury and may lead to improved assays that are more applicable for use in the clinical setting. In short, it remains to be determined if the characterization of these biomarkers could find practical applications.


Meloidogyne spp., Fusarium spp., Phytophthora spp., Abutilon theophrasti and Digitaria sanguinalis. Field trials found that DMDS + MNa (30 + 21 g a. i. m−2), both at a 50% reduced dose, effectively suppressed Meloidogyne spp. with a low root galling index (2.1% and 11.7%), significantly reduced the levels of Phytophthora and Fusarium spp. with a low root disease index (7.5% and 15.8%), gave very high cucumber yields (6.75 kg m−2 and 10.03 kg m−2), and increased income for cucumber growers with the highest economic benefits (20.91 ¥ m−2 and 23.58 ¥ m−2). The combination treatment provided similar results as MB standard dose treatment (40 g a. i. m−2) or DMDS standard dose treatment (60 g a. i. m−2) in pest control and yield, but was more effective than MNa standard dose treatment (42 g a. i. m−2). Usage of all chemical treatments gave better significant results than the untreated group of control. Considering the economic benefits, the DMDS plus MNa combination (30 + 21 g a. i. m−2) could be used for soil fumigation in cucumber production in China.Methyl bromide (MB), a dominant ozone-depleting substance, is scheduled to be completely phased out for soil fumigation by December 30th 2018, in China. The combined effects of dimethyl disulfide (DMDS) plus metham sodium (MNa) were assessed in controlling soilborne pests for soil fumigation. A study was designed in laboratory for the evaluation of the efficacy of DMDS + MNa to control major soilborne pests. At the same time, two trials were conducted in cucumber field located in Tongzhou (in 2012) and Shunyi (in 2013), respectively, in order to assess the potential of DMDS + MNa in controlling soilborne pests. Laboratory studies disclosed positive synergistic effects of almost all four used combinations on  Cucumis sativus L.) is a leading and important vegetable. Nematodes, soilborne fungi, oomycetes and weeds have a very great potential to lower yield in the protected cucumber production2. Methyl bromide (MB), an important soil fumigant, has widely been used to control soilborne pathogens, nematodes and weeds in areas of northern China. Though, MB must be completely phased out for soil fumigation by 30 December 2018 in China, because of its deleterious effects on stratospheric ozone3. The phasing out of the MB use as a pre-plant soil fumigant has greatly encouraged a deal of research aimed at finding economically acceptable, efficient, and simple alternatives4.In northern sites of China, cucumber  and calcium cyanamide are registered as MB chemical alternatives for cucumber in China. Chloropicrin (Pic)5, Dimethyl disulfide (DMDS)6, dazomet (DZ)7, and 1,3-dichloropropene 9 have been practiced as potential MB alternatives in cucumber. Though, there is no single chemical could replace MB totally10. To enhance the control activities for the pests of soilborne and broaden the action spectrum, the mixtures of promising alternative fumigants alone are becoming the scientific research hotspot in recent years. Several soil fumigant combinations, for example, 1,3-D/DZ7, 1,3-D/Pic11, DMDS/DZ12, 1,3-D/DMDS13 and 1,3-D/MNa14 have been tested to control soilborne pests for cucumber production.Currently, abamectin, fosthiazate, sulfuryl fluoride21, Pic and MNa21, 1,3-D and DZ22, Pic and DMDS20, 1,3-D and methyl isothiocyanate (MITC)23, Pic and methyl iodide (MeI)25 and so on as MB chemical alternatives for other crops production. Combinations of DMDS plus DZ have been reported as an efficient alternative to MB in Europe and China26. Both DZ and MNa are MITC generators. The combined use of DMDS and MNa to replace MB, therefore, was at least theoretically promising. Recently, the combined effects of DMDS and MNa were reported in muskmelon27 and cut flower28. Little information, however, was reported on combination of DMDS plus MNa in cucumber.Much information could also be retrieved in the literature on combinations of Pic and 1,3-DFusarium and Phytophthora spp.), nematodes (Meloidogyne spp.), and weed seeds  under laboratory experiment. Along with, 2 field trials were designed as well, for the determination of the MNa combinations and dimethyl disulfide as an alternative MB for cucumber yield in China at the same time.Current study was initiated to assess and confirm the efficacy of the combination of DMDS plus MNa on soilborne fungi and oomycetes (Meloidogyne spp. numbers by 90.7%. The maximum practiced dose in our study (80 + 40 mg a.i. kg−1) decreased the number of Meloidogyne spp. in terms of juveniles by 94.2%.All four applied rates of DMDS + MNa combination tested treatments showed a positive synergistic effect on root-knot nematodes Table , by decrPhytophthora and Fusarium spp.  decreased Fusarium spp. 93.6% and Phytophthora spp. 74.1%.Positive synergistic efficacy of four rates of DMDS + MNa tested was also observed on both p. Table . The infAbutilon theophrasti. However, the combination with the lower rate of MNa only showed synergistic efficacy (i.e. DMDS + MNa at 40 + 20 and 80 + 20 mg kg−1); while the combination with the maximum MNa rate did not exhibit synergistic efficacy (i.e. DMDS + MNa at 40 + 40 and 80 + 40 mg kg−1) ; the combination with the lower DMDS rate (i.e. DMDS + MNa at 40 + 20 and 40 + 40 mg kg−1) provided 70.8% and 95.9% control of Digitaria sanguinalis, respectively .The control groups at trial one in 2012) and trial two (in 2013) were found seriously occupied by 012 and tP = 0.05).The chemical treatments, except for MNa treatment, decreased the nematode infestation levels in trial one and trial two by 86.1% and 88.5%, respectively. The combination treatment of DMDS + MNa suppressed nematode levels by 86.1% and 89.7% in trial one and trial two, respectively; and both results statistically were not found different from MB or DMDS treatment, but higher significantly than MNa treatment at trial one  (Table The groups of control were seriously infested by both ls Table . The lev5) Table .Fusarium spp. levels were both provided by MB treatment at both trials, followed by the combination DMDS + MNa treatment, which decreased 93.6% Fusarium spp. and 80.0% at trial one and two.  (Table P = 0.05)  at both field trials (Table P = 0.05) (Table The 1st production of cucumber fruit was varied with fumigation treatments Table . Cucumbels Table . No sign5) Table .P = 0.05)  Table .Table 3EP = 0.05). The maximum root galling indexes were both noticed in the untreated plots . At trial one, the lowest index of root galling was provided by MB treatment, which was not different statistically from DMDS + MNa or DMDS alone, but observed lower significantly than MNa alone. At trial two, the minimum root galling index was also gained in MB treated plots, which did not change significantly from DMDS + MNa treatment but was lower significantly than DMDS alone or MNa alone (P = 0.05) . The maximum index for root disease (50.0% and 67.5%) were provided by the untreated control plots at two trials. At trial one, the lowest root disease index (3.3%) was provided by MB treatment, which was not different statistically from DMDS + MNa (7.5%), but was lower significant than DMDS or MNa alone. At trial two, the lowest root disease index (4.2%) was observed in treated MB plots, which was not significantly different from DMDS + MNa treatment or DMDS alone, but was observed lower than MNa alone, significantly (P = 0.05) . At trial one, treated plots with DMDS + MNa had the maximum production , which was not significantly different from the plots treated with MB  or DMDS alone , but was significantly higher than MNa alone . At trial two, treated MB plots produced the maximum yield , that was not found significantly different from the DMDS + MNa treatment  or DMDS alone , but significantly higher than MNa alone  (P = 0.05) . The plots treated with MB provided the highest gross incomes , which was not different significantly from the DMDS + MNa treatment  or DMDS alone , but significantly higher than MNa alone  (P = 0.05) , which was lower than MB treatment (4.07 ¥ m−2) or MNa alone (1.60 ¥ m−2) (Table P = 0.05)  of different soil fumigation treatments were assessed. Fumigation cost of different soil treatments were calculated by the sum of fumigants cost and film cost Table . The DMDreatment .07 ¥ m−2reatment .07 ¥ m−2reatment .07 ¥ m−229, which was confirmed in our both field trials. Anyhow, the overall formulations of DMDS exhibit poor efficacy against soilborne fungi and oomycetes, and moderate activity against weeds. So, additional herbicide and fungicide would be needed to improve soilborne fungi, oomycetes and weed control. MNa, a MITC producer, can control soilborne fungi, oomycetes and weeds efficiently. In the current study, it was observed that the MNa was comparatively less effective ton soilborne fungi and oomycetes than weeds. Thus, the combined use of DMDS and MNa as a promising soil fumigant is at least possible theoretically. All of the combinations of DMDS and MNa in laboratory studies exhibited positive synergy effects on root-knot nematodes, 2 major weed seeds and key soilborne fungi and oomycetes.To control root-knot nematode, DMDS is proven equally effective to MB not only in China but elsewhere28. DMDS is found to inhibit complex IV of mitochondrial electron transport chain (ETC), decrease the intracellular ATP concentration subsequently, which thereby activated neuronal KATP channels mediating membrane hyperpolarization and reduction of neuronal activity30. MITC as the active biocidal product of MNa is believed to react with amines and thiols in biological molecules31. It will be very difficult to assess a single mechanism of action that accounts for the synergism observed for the combination of DMDS plus MNa. However, two possible mechanisms may offer insight into the synergistic response of the fumigant combination32. One mechanism suggested is that sub-lethal exposure of soilborne pests to one fumigant may alter cell processes to such an extent that exposure to a second fumigant is more deleterious than would normally be expected33. Another suggested mechanism is that one fumigant causes changes in cell-wall permeability that increases the uptake or decreases the efflux of a second fumigant from the cell34. Further understanding of the activity of DMDS and MNa is necessary before the specific synergism mechanism can be deduced.The mechanism of specific synergistic activity of DMDS and MNa was still not clearly understood but the laboratory outcomes presented above, preliminarily indicated the possibility of their synergistic usage and affirmed the synergy activity of DMDS and MNa as previously reported by Gerik and Hanson−2 rate successfully inhibited Meloidogyne spp. root galling, decreased the colony-forming units (cfu) of Fusarium spp. sharply and Phytophthora spp. on media, sustained maximum production of cucumbers at the same time, and was not different significantly from 40 g m−2 MB dose or DMDS alone at 60 g m−2 dose, but improved than MNa alone at 42 g m−2 dose (P = 0.05). The results of field trials above confirmed that the combinations of DMDS and MNa are good choices than alone MB application. Furthermore, we compared the combination of DMDS plus MNa with the used soil fumigant combinations, such as 1,3-D/DZ, 1,3-D/Pic, DMDS/DZ, 1,3-D/DMDS and 1,3-D/MNa  are not available currently due to chemical incompatibility arising when combining them38. DMDS is highly volatile and a small molecule, so it diffuses and moves in the soil more quickly than MITC30. In this point, DMDS could make up the disadvantage of MITC and improve the control efficacy of the combination. Anyhow, uniform distribution in soil is needed by effectual mechanical injection or chemigation system. For better distribution of fumigant in soil, MNa is suitable to be applied by broadcast application or shank injection rather than chisel injection. If there was a good chemigation system available in the field, applying the fumigants by chemigation couldn’t be better.MNa requires relatively rigorous prerequisites just like DZ for the effective application. The aqueous solution of MNa is diluted by water and applied to the moist soil, where the compound rapidly decomposes to produce MITC. In the vapor and liquid phase of the soil, MITC is toxic to various soilborne pests. However, the physical and chemical properties of MITC  limit its movement and distribution in the soil following MNa applicationTrichoderma asperellum, Bacillus subtilis or others) are desirable before the DMDS and MNa combination can be suggested as an efficient substitute to MB for the yield of cucumber in China. The side effects of the combinations on soil beneficial organisms  should also be considered in further studies.In concluding remarks, the combination of DMDS and MNa fumigant applied by chemigation was almost equally to MB in terms of controlling knot-nematodes along with soilborne fungus while sustaining high cucumber yields and income for growers. However, much of the detailed work on its application type including its formulations and suitable composites with biological agents (eg. −1 soil. A soil sieve of 2-mm mesh was used and then mixing was done thoroughly. The moisture of soil was recorded 23.80% (w/w). Analysis for particle size were estimated through pipette method39. pH was determined in a 1:2.5 soil to water extract. Organic carbon contents were determined by wet oxidation followed the method of Walkley and Black40. Soil moisture contents were performed by remaining the soil at 105 ± 5 °C for 6 hours until constancy of mass was reached41.The combined and single efficacy of DMDS and MNa, was assessed under laboratory study. The collection of soil samples was taken from the 20 cm top in a cucumber field at Shunyi, Beijing, where nematodes and soilborne pests’ presence was abundant. The composition of soil sample was 20.34% sand, 76.25% silt, and 3.41% clay, with pH 6.40 and organic matter content 35.64 g kgAbutilon theophrasti seeds and fifteen Digitaria sanguinalis seeds were buried at 2 cm depth, respectively. The following fumigant treatments were performed with three replicates: DMDS alone , MNa alone , DMDS + MNa  and untreated group of control. DMDS or MNa was  injected into the soil by pipette, and then the desiccator was sealed immediately. The laboratory study photo  desiccators. In each desiccator, 10 see Fig. . was takIn years 2012 and 2013, two trials demonstration were performed in cucumber growing field at Tongzhou, Beijing and Shunyi, Beijing, respectively, where were both important production areas for cucumber. The farms, which have grown this vegetable for more than five years, are facing serious problems of soilborne pests including soilborne fungi, oomycetes, root-knot nematode, and other pests. Necessary details are given in Tables 99 DMDS TC , 42% MNa AS  and 98 MB TC  were selected in the current study. For soil mulch polyethylene film (PE)  was used.2 (3.2 m wide by 5.6 m long). A treatment as a reference of MB, DMDS alone, MNa alone, DMDS combination with MNa and untreated group of control were established. The application of DMDS was done through chemigation at 60 g a.i. m−2 rate; in order to increase the solubility of DMDS in the water, approximate 4% addition of Tween 80 is necessary13. MNa was practiced by chemigation at 42 g a.i. m−2 rate. Combined treatment of DMDS and MNa was practiced as follows: apply MNa by chemigation at 21 g a.i. m−2 rate, and then apply DMDS by chemigation 30 g a.i. m−2 rate. To cover the above treatments, PE film was placed. Application of MB between PE sheet and soil was through the hot gas method at 40 g m−2 rate. The fumigants were applied on 27 July 2012, and the tarps were removed on 17 August 2012. The cucumbers  were transplanted on 28 August 2012, and the final root disease investigation was conducted on 3 December 2012. In field trial two, the size of each plot was 20.16 m2 (3.6 m wide by 5.6 m long). All of the treatments in field trial two were the same as those in field trial one  g4. Cucumber root disease severity was also rated on a scale of 0−4, 0 = healthy plant and root, without disease; 1 = black brown rotten roots comprise < 25% of the entire root system; 2 = 26−50%; 3 = 51−75%; and 4 = 76−100% black brown rotten roots45. Cucumber fruit yields and income of growers were collected after every harvest and summed together at the end of the trials.Height of cucumber plant was determined at 4 weeks after transplant (WAT) (per plot 20 plants). Calculation of root galls and the severity of cucumber root disease were done at the end of the trials (per plot 20 plants). Plants were rated on a scale of 0–4: 0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100% roots galledX is % nematode mortality, N1 is the number of dead nematodes, and N2 is the number of the live nematodes.Mortality of nematode was calculated by the equation following.Y is the % corrected nematode mortality, while X1 is the % nematode mortality of fumigated treatments, and X2 is the % nematode mortality of untreated control.Corrected mortality of nematode was considered by the equation following.Y is the fungi or weed seeds control efficacy, X1 is the fungal populations or weed height in untreated control group, X2 is the fungal populations or weed height in treated fumigated plots.Controlling fungi or weed seeds efficacy was measured according to the equation following.et al.46.E0 is the expected control efficacy of DMDS plus MNa, X1 is the actual measured control efficacy of DMDS, X2 is the actual measured control efficacy of MNa. E is the actual measured control efficacy of DMDS plus MNa. If E > E0, the combination of DMDS plus MNa was synergistic; if E < E0, the combination of DMDS plus MNa was antagonistic.The DMDS plus MNa control efficacy was calculated by the equation following, using methods described by Limpel The effectiveness of controlling root-knot nematode or soilborne fungi or oomycetes was measured according to the Eq. .RGI) or root disease indices (% RDI) using the formula described by McKinney45:v = class value, N = number of observed plants, and X = highest value of the evaluation scale.Root galls scores or disease scores recorded for each plot were respectively converted into root galling index  was run to conduct analysis of variance (ANOVA) for data analysis. Data for populations of soilborne fungus and knot-nematode were transformed as necessary , however, all of the data reported here as non-transformed values. Fisher’s LSD test at Supplementary Information


The link between autistic people having a mind that is difficult to read  and being perceived unfavorably was investigated. Videoed Autistic and neurotypical targets from Sheppard et al. (PLOS ONE 7(11):e49859, 2016) were scored for how readable they were when reacting to a distinctive greeting from the experimenter. These videos were presented to new groups of perceivers  who rated neurotypical targets more socially favorably than autistic targets irrespective of whether details of the experimenter’s greeting were concealed (Study 1) or disclosed (Study 2). Target readability correlated with ratings of target favorability (r = .58 and r = .63), independent of target diagnosis. Perceivers might rate targets unfavorably because they experience difficulty reading them, though other interpretations of the correlation are also possible. Much research has investigated how effectively autistic people read others’ minds, and some results suggest poor ability and developmental delay in this arena (Baron-Cohen Two recent studies report that people (perceivers) find it difficult to read autistic individuals (targets). The most recent  are described in Sheppard et al. . BrieflyAs described in Sheppard et al. , a Sony M = 24.5 years, SD = 1.98) were recruited through the ‘participant recruitment system’ and advertisements at the University of Nottingham. Although these perceivers were older than the targets on average  aged between 20 and 28 . All 40 target videos were shown to each perceiver on a 15 in. MacBook Air, presented in random order using PsychoPy2 version 1.85.2 Peirce . Each peQuestions were adapted from Sasson et al. . Due to Perceivers rated each target on nine social favorability scales. We averaged ratings across all perceivers for each of these nine scales and with each target group. Mean ratings appear in Fig. r > − 0.75, p < .001). A global ‘social favorability’ variable was derived from the sum of the averages of all nine scales . This was justified given the high correlations between all measures, supported by the very high Cronbach’s alpha of .98, indicating strong internal reliability of the nine scales. A 2 × 4 repeated measures ANOVA was conducted to assess the associations between group and scenario type on global social favorability. A main effect of target group was found, F = 104.14, p < .001, with autistic targets viewed less socially favorable than non-autistic targets. A main effect of scenario was also found, Greenhouse–Geisser corrected due to violation of sphericity, F = 86.57, p < .001. Post-hoc pairwise comparisons were conducted between the four scenario types, with Bonferroni correction: the main effect of scenario was driven by targets in the waiting scenario being rated significantly less socially favorable than those in all other scenarios (p < .001); there were no differences between the other three scenarios in how socially favorable targets were rated.Bivariate Pearson correlations revealed a strong positive correlation among all nine social favorability factors Table . ‘AwkwarF = 29.65, p < .001, raising the possibility that perceiver ratings were more positive for one target group over the other, depending on the scenario. To explore this possibility, four post hoc paired-samples t tests were employed to compare participant performance across the two target groups for each scenario independently. Autistic targets were perceived less socially favorable than typically developing targets in the compliment scenario t(30) = 9.64, p < .001, Joke scenario t(30) = 4.08, p < .001, and story scenario t(30) = 8.11, p < .001. However, there was no significant difference in how socially favorable target groups were rated in the waiting scenario, t(30) = 0.44, p = .66, where both groups were rated rather negatively. Reported p-values are following Bonferroni correction. Summing up, it is notable that perceiver ratings of social favorability effectively discriminated between autistic and typically developing targets, even though perceivers were not given any information that the targets formed two discrete groups according to clinical diagnosis.A significant interaction effect Fig.  between F = 6.56, p = .015, confirming that autistic targets were less readable than non-autistic targets. A main effect of scenario was also found, F = 17.467, p < .001, but the interaction term was nonsignificant, F = 1.631, p = .202. Bonferroni corrected pairwise comparisons revealed that readability in the waiting scenario was significantly greater than in the other scenarios, as illustrated in Fig. Were autistic targets less readable than non-autistic targets? We conducted a confirmatory analysis by repurposing archived readability data from Sheppard et al.  and calcF1,2 = 6.56,r = 0.58, p < .001, see Fig. r = 0.45, p < .01. It seems the relationship between target social favorability and target readability is not a simple function of autistic targets being unreadable and also being perceived as rather less socially favorable. Perhaps the relation between readability and social favorability is more fundamental in a way that transcends target clinical status.Was the tendency for autistic targets to be perceived less positively associated with their being less readable? To begin to answer this question we conducted a bivariate correlation between target readability and target social favorability across three scenarios (excluding the waiting scenario). The waiting scenario was excluded because this condition yielded high readability perhaps because targets were merely waiting while the experimenter was apparently preoccupied with something else, whereas in the other scenarios the experimenter was actively engaging with the target. Hence, perceivers might easily identify this scenario through a process of elimination. Also, favorability ratings were quite different (much more negative) than for the other scenarios, irrespective of diagnosis, perhaps because targets appeared disagreeable on being neglected by the experimenter. Based on the remaining three scenarios , we found a positive relationship between target social favorability and target readability, The second study offers a replication but with disclosure to perceivers of the particulars of the scenario each target was experiencing. Such information might help perceivers to contextualize target behavior, which should reduce noise in mean target favorability data. Accordingly, we might expect the correlation between target readability and favorability to be even stronger than in the first study. We might also find targets in the waiting scenario are no longer rated less favorably than in other scenarios when perceivers can interpret apparently disagreeable target behavior arising as a product of a disagreeable situation.M = 19.8 years, SD = 1.94) took part; none had participated in Study 1. All were native English speakers.Thirty perceivers  aged between 18 and 25 . In this study perceivers were informed which scenario each target was experiencing, and the name of each scenario appeared before each target video for 5 s.F = 127.94, p < .001. A main effect of scenario was also found, F = 66.80, p < .001, Greenhouse–Geisser corrected for violation of sphericity. Post-hoc pairwise comparisons with Bonferroni correction revealed that targets in the waiting scenario were rated significantly less socially favorable than targets in other scenarios (p < .001); there were no differences between the other three scenarios in how socially favorable targets were rated.Group differences across the nine ratings of social favorability are apparent in Fig. F = 29.1, p < .001, raising the possibility that ratings were more positive for one target group than the other, depending on the scenario. To explore this possibility, four post hoc paired-samples t tests were employed to compare perceiver ratings across the two target groups for each scenario independently. Autistic targets were rated less socially favorable for the compliment scenario t(29) = − 10.26, p < .001, Joke scenario t(29) = − 4.75, p < .001, story scenario t(29) = − 6.60, p < .001 and waiting scenario, t(29) = − 3.98, p < .001. Reported p-values are following Bonferroni correction. To further explore the interaction, repeated measures ANOVAs were conducted to determine whether there was an effect of scenario for each group. For the autistic group, a main effect of scenario was found, F  = 39.39, p < .001, Greenhouse–Geisser corrected. Post-hoc pairwise comparisons indicate that targets were rated less favorably in the compliment and waiting scenarios compared with all other scenarios, p < .05, but there was no difference between the story and joke scenarios, p = .79. For the non-autistic group, a main effect of scenario was found, F = 65.90, p < .001. Post-hoc pairwise comparisons showed that targets in the waiting scenario were rated as less socially favorable than those in the other three scenarios, p < .001.A significant interaction effect Fig.  between r = 0.63, p < .001. A partial correlation controlling for target group (autistic or non-autistic) confirmed that the relationship between social favorability and readability survived, r = 0.52, p = .004. Hence, it seems the correlation is not driven by autistic targets being unreadable and being perceived unfavorably, while typical targets are readable and also perceived favorably. As with Study 1, the significant relationship transcends clinical status.A bivariate correlation was conducted between target readability  and not just how they look, influences perceiver ratings. This was most notable in the waiting scenario where targets appeared disagreeable because the experimenter was behaving rudely and perceivers attributed disagreeable target dispositions on witnessing negative target signals. Even when the scenario was disclosed, perceivers persisted in rating targets negatively in this scenario.When information about the target’s situation was concealed, non-autistic and autistic targets were perceived equally negatively in the waiting scenario. This is a striking finding given that autistic people have been judged more negatively across various other circumstances, including the other scenarios within this study and in previous research  and where it was disclosed (Study 2). This suggests it made no difference to perceivers’ tendency to rate non-autistic targets more favorably than autistic targets whether or not they (perceivers) had some contextual information to assist in explaining target behavior. The association between readability and favorability survived even when clinical diagnosis was partialled out, raising the possibility that the condition of autism is not driving this association.The statistical association between readability and perceived social favorability does not illuminate the direction of causality or whether the association is the product of a mediating variable. At least, though, we are able to eliminate target diagnostic status as such a mediator. Determining causality is a matter for future research, where larger data sets captured at multiple time points might enable statistical modelling of the directional influence of one variable (e.g. readability) over the other (e.g. favorability).reward explanation and a trait explanation. Anders et al.  and also result in more negative social favorability judgments (in favorability studies). In short, perhaps autistic people behave in a way that is perceived by non-autistic people as being ‘out of the ordinary’, rendering them unreadable and also causing them to be perceived unfavorably. Here, ‘out of the ordinary’ is a mediating variable that explains the correlation between unreadability and perceived unfavorability without needing to posit a direct causal link between the two variables.A further possibility is that some other variable mediates the relationship between readability and social favorability. In order for the target’s readability to directly influence how socially favorable they are perceived (either via a reward or trait route), this would require perceivers first to make some kind of judgment about how readable the target they are viewing actually is. This might happen in Study 2, where perceivers were told the situation to which the target is reacting: perceivers may notice a mis-match between a target’s behavior and expected behavior in that scenario and infer that they are unable to read the target. It is less apparent how perceivers could infer readability in Study 1, where no contextual information was provided. Perhaps the behavior of some targets is unusual in The possibility that behavior is perceived as being ‘out of the ordinary’ leads to suggestions for future research into why autistic people are difficult to read; and at least two rival explanations are worth considering. One possibility raised here is that the behavior of autistic people is rather different from that of neurotypical people irrespective of context, as would be the case if autistic people had flattened affect and thus were inexpressive such that the signal in their behavior was weakened. However, we already know this is not true of the targets who served in this study (Sheppard et al. In future research, if each target experiences a variety of scenarios, it will be possible to determine if autistic people emit the same stereotyped response to all scenarios. If they do so, this would lend support to the suggestion that autistic people behave in a way that is ‘out of the ordinary’ in social contexts. In this case, we would expect perceiver judgments to be around chance level or at best only slightly above chance level. In contrast, if autistic participants emit misleading signals, then perceivers will make systematic errors, in which case accuracy in inferring the scenario will not be at chance level but will be significantly below chance level.If being unreadable is associated with being rated socially unfavorable, as suggested by our findings, it could in turn have very negative consequences for the development of autistic individuals. This follows if being perceived unfavorably is a barrier to inclusion in the social world, where autistic people, who are in the population minority, instead are condemned to isolation. The social arena provides the experiences essential to understanding how other people behave, think and feel. In being excluded from such experiences, autistic people are trapped in a ‘vicious circle,’ lagging further and further behind in their developing understanding of the psychology of other people, making it ever more difficult for them to be accepted and included Mitchell . This un


Joubert syndrome  is a recessive developmental disorder characterized by cerebellar vermis hypoplasia and a distinctive mid‐hindbrain malformation called the “molar tooth sign” on axial magnetic resonance imaging. To date, more than 35 ciliary genes have been identified as the causative genes of JS.CEP104, OMIM: 616690).Whole exome sequencing was performed to detect the causative gene mutations in a Chinese patient with JS followed by Sanger sequencing. RT‐PCR and Sanger sequencing were used to confirm the abnormal transcript of centrosomal protein 104  (GenBank: NM_014704.3) and consistent with the autosomal recessive inheritance mode.We identified two novel heterozygous mutations of CEP104 mutations, which expands the mutation spectrum of CEP104 and elucidates the clinical heterogeneity of JS.Our study reported the fourth case of JS patients with  CEP104 in the proband, which were c.2364+1G>A and c.414delC (p.Asn138LysfsTer11). (c) This is the fourth case of JS patients with CEP104 mutations, which expands the mutation spectrum of CEP104 and elucidates the clinical heterogeneity of JS.(a) We report on a Chinese patient diagnosed with Joubert syndrome (JS), presenting developmental delay, hypotonia, oculomotor apraxia, and molar tooth sign. (b) Whole exome sequencing identified two novel heterozygous mutations of  We performed the whole exome sequencing using the proband's DNA. In brief, the whole exome library was prepared using Agilent SureSelect Human All Exon V6 kit (Agilent Technologies Inc.) according to manufacturer's standard protocol. Sequencing was performed on Illumina Novaseq 6000 platform (Illumina Inc.) with 150 bp paired‐end reads. Reads were aligned to reference genome hg19 (GRCh37) by Burrows‐Wheeler Aligner (v.0.7.17) along with Samtools. PCR duplicates were removed by Picard tools (V2.18.4). Variations were called using GATK  and annotated with Ensembl Variant Effect Predictor (v91.3)  following the manufacturer's protocol for variants validation in the proband and his parents. Primer 4F (5′‐CTGTTGATCCTGCATAGGGG‐3′) and primer 4R (5′‐TCAGCAGTCTCCCAGAAGAGAT‐3′) were used for exon 4 amplification, while primer 18F (5′‐TTTCAGGCACCTCCTTGGTG‐3′) and 18R (5′‐TATGGAATGACTCGCACGCA‐3′) were used for exon 18 amplification.2.3™ Blood RNA Tube . Tempus™ Spin RNA Isolation Kit  was used for RNA extraction from whole blood cells of the proband and his parents. One microgram of RNA was reverse transcribed into cDNA using SuperScript™ IV First‐Strand Synthesis System Kit . The primer sequences used for cDNA amplification were 5′‐GAATCAGGACATTCAAGGAGGGA‐3′  and 5′‐TTTCCATGCCTCTTCTCCAGG‐3′ . The PCR amplification products were analyzed by agarose gel electrophoresis. The purified DNA bands were cloned into pMD19‐T Vector (TaKaRa) and validated by Sanger sequencing.The blood samples of proband and his parents were collected using Tempus33.1The patient is a 3‐year‐old boy, who is presented with hypotonia and psychomotor developmental delay. He is the second child of an unrelated couple without personal or familial medical history. Born by caesarean section at 39 week gestation, the patient showed normal birth measurements: weight of 3.05 kg and height of 48 cm. On examination, he has low‐set ears, epicanthus, and strabismus. Hypotonia and psychomotor developmental delay was obvious in the patient: he was unable to hold his neck until 10 months and sit unaided at 12 months, and he cannot stand or walk independently by 3 years. He presented with speech delay and can only speak monosyllabic words. The developmental quotients of motor, object, adaptability, language, and social abilities were evaluated by Gesell Developmental Schedules (GDS). Mild‐to‐severe retardation of the GDS, especially language and motor abilities, was observed. Brain magnetic resonance imaging (MRI) showed cerebellar vermis dysplasia, thickened and elongated superior cerebellar peduncles and MTS  (c.414delC [p.Asn138Lysfs*11] and c.2364+1G>A) were found in the proband , which was c.6012‐2A>G, but no other susceptive pathogenic variant in CEP290 was found.Compound heterozygous variants in d Figure . This vad Figure . Of note3.3CEP104, spanning exon amplification product was analyzed by agarose gel electrophoresis. Four bands were observed in the PCR products of the samples from the proband and his father, while only one band (400 bp) was detected in the products from his mother, brother and the healthy control  mutations  in the proband which are the cause of the disease. Four pathogenic variants were previously reported in three patients from different families, including two splicing site mutations, one nonsense mutation and one frameshift mutation  (Bachmann‐Gagescu et al., Exome sequencing revealed two novel compound heterozygous variants of CEP104 was identified as a ciliary tip protein by a comparative, quantitative proteomic study in Chlamydomonas (Satish Tammana, Tammana, Diener, & Rosenbaum, CEP104 caused ciliogenesis defect in both Chlamydomonas and human RPE‐1 cells (Satish Tammana et al., CEP104 was a multidomain protein and interacted with several cilia and microtubule‐related proteins, including CP110, CEP97, end‐binding protein, and tubulin (Al‐Jassar et al., CEP104 (Al‐Jassar et al., CEP104 with CP110 (Al‐Jassar et al., CEP104, which expands the mutation spectrum of CEP104 and elucidates the clinical heterogeneity of JS. Future description of other patients with mutations in CEP104 and the following studies of their underlying cell biology and physiology will define new mechanisms on the role of CEP104 and cilia in brain development, especially JS.In summary, this JS patient has two novel mutations in http://www.1000genomes.org/1000 Genomes ://exac.broadinstitute.org/ExAC http://gnomad.broadinstitute.orggnomAD ://www.ncbi.nlm.nih.gov/SNP/dbSNP ://omim.org/OMIM None declared.


Dienogest has been shown to substantially improve endometriosis-associated symptoms such as debilitating chronic pelvic pain, and in turn, health-related quality of life (HRQoL). To date, there is no data on patient-reported outcomes reflecting the real-world practice in Asia where endometriosis is a relevant health, social and economic burden. This non-interventional, multi-center, prospective study aims to investigate the influence of dienogest on HRQoL.Asian women received dienogest (2 mg/daily) and were followed for 24 months. The effectiveness of dienogest to improve HRQoL and endometriosis-associated pelvic pain (EAPP) was assessed by patient-reported outcomes. HRQoL, especially the “pain” domain as primary endpoint, was evaluated with the Endometriosis Health Profile-30 (EHP-30) questionnaire. The numeric rating scale served to determine changes in the severity of EAPP. Within the presented interim analysis (data cut-off: 2017-11-27), the mean changes in EHP-30 and EAPP scores from baseline to 6 months upon availability of the data were evaluated. Treatment-emergent adverse events (TEAEs) and bleeding profiles were documented.Dienogest therapy decreased EHP-30 scores in all assessed domains . Primarily, the “pain” domain was improved in 78.4% of patients. EAPP was reduced , highlighted by a mean reduction of the pain score by − 4.5 points. Patients with a higher EAPP score at baseline had an increased response to dienogest (− 6.2 points mean change) compared to patients with low baseline EAPP severity (− 1.4 points mean change). Both surgically and clinically diagnosed patients described comparable pain reduction, as well as women with or without prior treatment.Drug-related TEAEs were documented for 31.5% of patients, with amenorrhoea (5.9%) and metrorrhagia (5.1%) being the most common events. The bleeding pattern was changed upon dienogest, characterized by decreased normal bleeding (84.2 to 28.8%) and increased amenorrhea (3.2 to 42.9%) at 6 months.The data indicate an amelioration of HRQoL and EAPP upon dienogest therapy. No new safety signals were observed. Therefore, its use as first-line therapy for long-term management of debilitating and chronic endometriosis-associated pain represents an interesting option that remains to be further investigated.NCT02425462 Registration date: 2015-04-24. Registration timing: prospective.Name of registry: Clinical Trials Clinicaltrials.gov registration number:  Endometriosis is a chronic disease that affects approximately 10% of all women in reproductive age and up to 50% of infertile women . CurrentEndometriosis is characterized by endometrial-like tissue outside the uterus. Conventionally, diagnosis of endometriosis required the combination of laparoscopic inspection of the pelvis with histological verification of endometrial glands, but this recommendation is not supported by robust evidence –8.Guidelines recommend a non-invasive clinical diagnosis based on clinical symptoms and patients history , 6, 7. HClassic endometriosis symptoms are chronic pelvic pain, dysmenorrhea, dyspareunia and infertility; furthermore, endometriosis can cause symptoms arising from other organs involved, such as dyschezia, tenesmus and dysuria and/or hematuria . QualityTo date, the impact of dienogest treatment on quality of life has not been extensively studied in the real-life setting. Moreover, most of the clinical trials on dienogest enrolled only women with definite surgical diagnosis of endometriosis. This international non-interventional, non-controlled, multi-center, prospective cohort study (ENVISIOeN) aims to assess the effectiveness of dienogest  in improving HRQoL in clinically and surgically diagnosed Asian women with endometriosis under routine clinical practice. The interim analysis evaluated the pain domain of HRQoL as primary goal, as well as EAPP and safety as secondary goals after 6 months of follow up.Clinicaltrials.gov (NCT02425462).The study was carried out at 36 sites in Thailand (five centres), Indonesia (ten centres), Republic of Korea (twelve centres), Malaysia (four centres), Philippines (three centres) and Singapore (two centres) in accordance with the amended version of the Declaration of Helsinki (Oct 2013) and complied with Good Clinical Practice. For more information, see The inclusion criteria were: Asian female ≥18 years old; clinical or surgical diagnosis of endometriosis; presence of EAPP; uninfluenced decision taken by the physician to newly prescribe dienogest; written informed consent. Women were excluded in case of participation in an investigational program with interventions outside of routine clinical practice; any contraindication listed in the local summary of product characteristics. All eligible patients were treated according to standard medical guidelines or usual care of the participating institute.The observation period of 24 months for each patient enrolled in this study consisted of the treatment phase and, in case of discontinuation of the treatment, the follow up period. Subjects were followed whether or not they remained on the treatment with dienogest. Information on the date and reasons for discontinuation were collected.The primary endpoint was to assess the effectiveness of dienogest to change HRQoL with special attention to the dimension of pain. Patients were asked to complete the Endometriosis Health Profile-30 (EHP-30) at baseline and at the visits 6 months after start of treatment. The EHP-30 is a disease-specific, reliable and valid instrument to measure the effects of endometriosis on HRQoL, especially on physical, psychologic and social aspects, from the patients’ perspective, as described previously –19. SecoAll TEAEs, including the ones leading to discontinuation were recorded at each visit throughout the study and summarized using the MedDRA coding system (not to be confused with the bleeding profile terminology). The bleeding profile was evaluated based on the following categories (definition within the figure): normal bleeding, irregular bleeding cycle, amenorrhea, intermenstrual bleeding/spotting. Further secondary endpoints analyzed within the scope of the interim analysis were continuation rate as well as patients’ and physicians’ satisfaction rate.The treating physician collected historical data  from medical records if available and treatment-related data during visits that took place in routine practice.To circumvent the general bias susceptibility inherent to observational studies, e.g. selection bias, it ensured that the study population represented routine patient profiles treated with dienogest in Asia. The patients were selected only based on inclusion and exclusion criteria in a consecutive manner.The calculation of the sample size focused on the pain dimension in the EHP-30 score. Assumptions for the sample size have been based on earlier studies that collected information on quality of life in patients with endometriosis who have been treated daily with a progestin . For thiIn terms of safety this study was sufficiently large to capture uncommon adverse events. With the planned sample size at least one adverse event with a relative frequency of 0.3% was expected to be observed with a probability of 90%.Effectiveness analyses were conducted on the efficacy analysis set (EFF) that included all patients with evaluable EHP-30 questionnaire at baseline and at least one evaluable EHP-30 questionnaire post-baseline between week 12 and 36 after start of treatment. Measurements after discontinuation of treatment were excluded. Safety analyses were performed on the full analysis set (FAS) and comprised all patients that took at least one dose of dienogest.The HRQoL parameters were assessed as described previously. The score ranged between 0  and 100  . For EAPThe analysis was primarily of explorative and descriptive nature. All variables were analyzed descriptively with appropriate statistical methods. SAS release 9.4  was used for data analysis. Continuous variables were described by visit (baseline and 6-months) and as mean change from baseline to 6 months for each item of the questionnaire, if applicable. For change from baseline in EHP-30 scores descriptive statistics as well as confidence intervals have been provided. Frequency distributions of patients with improvement, deterioration or no change in EHP-30 and EAPP scores were summarized. Missing data was not replaced and was given as “missing” in the tables. All adverse events were presented within incidence tables as preferred terms by system organ class. The descriptive analysis of adverse events was the basis to assess tolerability and safety of dienogest in the study population. The analyses described were performed on TEAEs. Non TEAEs were tabulated without further stratification. Partially missing TEAE onset dates were imputed following a worst case approach. The earliest possible date was used (as described above), but in case the AE onset could be before or after start of dienogest, TEAE onset was imputed by the first day of dienogest treatment.n = 97 evaluable patients; EFF). Leading reasons for end of observation were patient lost to follow-up  and regular end of study .Overall, a total of 895 patients were enrolled at 36 sites from 04/2015 to 08/2016. The FAS comprised 865 patients who were eligible, gave their informed consent and took at least one dose of dienogest. Furthermore, 510 patients with evaluable primary outcome were included into the EFF. The mean ± SD duration of treatment with end of observation was 14.1 ± 7.5 months  and chronic pelvic pain . While endometriosis was diagnosed within 1 year before initial visit in the majority of the patients , only less than half of the women reported onset of first symptoms within this time span . Moreover, 29.9% of women (n = 259/865) documented appearance of first symptoms between one and 5 years before and 23.0% (n = 199/865) more than 5 years before. In fact, 24.6% of patients diagnosed within 1 year before initial visit experienced their first symptoms 1–5 years earlier and 12.4% of patients even > 5 years before, amounting to approximately 37% of patients with delayed diagnosis of endometriosis. The time period from appearance of first symptoms to first diagnosis tended to be shorter in surgically diagnosed patients than in patients with clinical only diagnosis . The majority of women  underwent prior surgery to treat endometriosis-associated pain. The proportion of women with pain recurrence despite surgical treatment was 27.4% (n = 123/449) and the mean ± SD duration until pain recurrence after surgery was 19.3 ± 21.0 months . A total of 179 women received previous hormonal treatment, with 56.4% of those women (n = 101/179) suffering from pain recurrence after a mean ± SD time of 9.9 ± 13.9 months . Pain medications were given to 89 patients , predominantly 2–3 days per week , with a mean ± SD pain recurrence after 11.9 ± 37.2 days  after or during pain therapy.Baseline characteristics are summarized in Table n = 348), whereas 14.0% (n = 62) showed no change and 7.7% (n = 34) reported deterioration. In the control domain, 70.5% (n = 313) of women experienced an improvement, whereas 18.2% (n = 81) and 10.8% (n = 48/444) had no change or deterioration, respectively. In the domains of emotional well-being, social support and self-image, 61.3% (n = 272), 55.4% (n = 256) and 42.1% (n = 187) of patients reported improvement, respectively.Table n = 440). In the domain of work, the mean change ± SD was − 21.8 ± 25.3. Mean changes ± SD in other domains were as follows: − 19.5 ± 29.8 (relationship with children), − 11.4 ± 25.2 , − 7.2 ± 22.2 (treatment) and − 8.6 ± 21.7 (infertility).Baseline and 6-months visit EHP-30 scores as well as changes within the modular questionnaire are summarized in Table n = 434). The total study population reported a mean change ± SD of − 4.5 ± 3.0 (Table n = 402). Remarkably, 84.3% (n = 366) of the total patient population described an improvement of EAPP with dienogest therapy, whereas 5.5% (n = 24) and 2.8% (n = 12) reported no change or even deterioration, respectively  of women with EAPP baseline severity ≤4 experienced an amelioration of EAPP  compared to women with baseline severity ≤4  and − 5.1 ± 3.2 (n = 97) for surgically and clinically only diagnosed patients, respectively, were comparable to the mean change of the total population (Table n = 279/332) in surgically diagnosed patients vs. 85.3% (n = 87/102) in clinically only diagnosed patients  and consequent improvement rate of 93.0% (n = 40/43) marginally exceeded the mean change of the total population. Finally, the previous surgical or hormonal treatment did not affect the extent of EAPP change following dienogest administration  to 28.8% (n = 187/865) after 6 months, whereas amenorrhea increased from 3.2% (n = 28/865) to 42.9% (n = 279/865), as displayed in Fig. With regard to the bleeding profile of the patients, normal bleeding was decreased from 84.2% (n = 322/488) and physicians  were satisfied with the therapy. Perceivable overall symptom improvement was reported by 52.1% (n = 254/488) of cases. Finally, the majority of patients continued treatment with dienogest  after 6 months.A large proportion of patients  discontinued dienogest therapy due to drug-related TEAEs. Abnormal uterine bleeding  was the most prominent reason for discontinuation . TEAEs irrespective of causality were documented in 35.5% of patients (n = 307/865) and were predominantly of mild-to-moderate intensity. Most common TEAEs are presented in Table In total, there were nine serious adverse events (SAEs) reported in eight patients Table . ThereofThe debilitating, chronic and relapsing nature of endometriosis points to an unmet need for effective treatment approaches . A studyAccordingly, our data suggest that dienogest might represent an option in the treatment of endometriosis in Asian women in the real-life setting, particularly in the improvement of HRQoL. As such, nominal improvements throughout all domains of the EHP-30 questionnaire after 6 months compared to baseline were observed. The most striking change was documented in the domain of pain, where an improvement in 78.4% of patients after 6 months was documented. Importantly, the changes in the other core scores were in line with changes in the domains of the modular questionnaire, underscoring the relevance to relieve pain for the improvement of all aspects of HRQoL. As some aspects of QoL such as social support and self-image might be affected through other psychosocial factors than EAPP, these domains appeared to be less influenced by dienogest. With regard to the EAPP, dienogest therapy was associated with a change of pain after 6 months, and an improvement rate of 84.3%. Women with higher EAPP score > 4 at baseline visit described greater improvement (92.5%) at 6-months visit, in contrast to 71.3% improvement in women with low baseline severity of EAPP ≤4.According to recent guidelines, endometriosis should be classified with questions about pelvic pain and infertility in low resource settings in order to prescribe the most successful treatment possible prior to or even without definite surgical/histological confirmation . Thus, eThe bleeding profile was affected by dienogest therapy, with decreased normal bleeding (28.8%) and increased amenorrhea (42.9%) after 6 months. Commonly, abnormal uterine bleeding is a well-known adverse event upon long-term treatment with progestins. A pooled analysis of clinical trials revealed that amenorrhea increased from < 5 to 30% upon extended treatment duration, whereas undesirable patterns such as abnormal, frequent or prolonged bleeding decreased –16, 24. This international non-interventional study design allows observing dienogest use among different Asian countries under real-world clinical settings for the first time. Therefore, all decisions in terms of diagnostic procedures and management of the disease are fully dependent on mutual agreement between the patient and the treating physician, without interference by a sponsor. As discussed above, there is an unmet need to improve the impaired quality of life of women with endometriosis, highlighting the clinical relevance of the study. A potential limitation of this study was the low number of evaluable patients included in the EFF, resulting from a lower feasibility of regular participation for many patients in the real-world setting. The local conditions at the participating sites possibly hampered the women to comply with the scheduled time points. Furthermore, there were certainly differences between the healthcare systems of the participant countries, in particular in terms of diagnosis and treatment. The major limitation of this study however is that within a single arm design lacking appropriate controls, the effects on HRQoL and safety cannot be fully attributed to the dienogest therapy. Other unknown factors might be underestimated and therefore, effectiveness and safety of dienogest can only be interpreted to a limited extent and requires further investigation.In conclusion, our data indicated that therapy with dienogest could improve HRQoL and change the perception of EAPP in Asian women suffering from debilitating endometriosis symptoms. As no new safety signals were observed and a good satisfaction rate and compliance was achieved, dienogest might be an interesting therapeutic option for the long-term management of endometriosis among Asian women.


Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients.We characterized androgen receptor‐mediated tissue factor regulation in prostate epithelial cells.Androgen receptor is dampening tissue factor expression in prostate epithelial cells.Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates.ADT), which is aimed at reducing circulating testosterone levels to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the most important mediators of coagulation and VTE, but, so far, there are limited data on androgen receptor (AR)‐mediated TF gene expression.Prostate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (AR‐mediated TF regulation in vitro and in vivo.To characterize LNCaP and MyC‐CaP to test whether TF expression is regulated by AR. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector to identify the transcription factor‐binding sites that mediate TF regulation downstream of AR. Finally, we used castration experiments in mice to characterize AR‐mediated TF regulation in vivo.We used the androgen‐dependent prostate cancer cell lines TF is directly regulated by AR. In LNCaP cells, nuclear factor‐κB signaling and EGR1 mediate TF expression. By using castration experiments in mice, we could detect upregulation of TF and early growth response protein 1 mRNA and protein expression in prostate epithelial cells.AR is crucial for dampening TF expression, which could be important for increased TF expression and TF‐positive microvesicle release in androgen‐deprived prostate cancer patients. In 2018, ∼ 164 690 new cases of prostate cancer will be diagnosed in the USA, which makes it the second most diagnosed cancer after lung cancer Prostate cancer is a known risk factor for venous thromboembolism (VTE), which includes deep vein thrombosis and pulmonary embolism One of the most important mediators of coagulation is tissue factor (TF). TF is a transmembrane glycoprotein that has important functions in blood clotting and hemostasis, but also in tumor progression and metastasis In prostate tumors, TF expression appears to be confined to malignant luminal epithelial cells in vivo.Despite a potential role for TF in prostate cancer, an analysis of androgen receptor (AR)‐mediated regulation of TF expression has so far not been reported. We show that TF expression and activity are regulated after dihydrotestosterone (DHT) treatment in the androgen‐dependent prostate cancer cell lines LNCaP and MyC‐CaP. This DHT‐mediated TF regulation is AR‐dependent, as it could be blocked by addition of the AR antagonist bicalutamide. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector and analyzed the contribution of known transcription factor‐binding sites to AR‐mediated TF regulation. Using this approach, we found that TF regulation is mediated through NF‐κB and EGR1 transcription factor‐binding sites in LNCaP cells. Finally, by using castration experiments in mice, we provide evidence that an AR–NF‐κB–EGR1 signaling axis is also operative in prostate epithelial cells LNCaP and MyC‐CaP cells were purchased from ATTC. PC3 and DU145 cell lines were a kind gift from M. Unseld . LNCaP, PC3 and DU145 cells were routinely maintained in normal RPMI medium containing 10% fetal bovine serum (FBS), phenol red, and antibiotics. MyC‐CaP cells were routinely maintained in normal Dulbecco's modified Eagle's medium (DMEM). For DHT stimulation experiments, LNCaP, MyC‐CaPs, PC3 and DU145 cells were seeded at 50–60% density in RPMI medium or DMEM, without phenol red, containing 5% charcoal‐stripped FBS . Transient transfections of all reporter and expression plasmids were performed with Turbofec , according to the manufacturer's instructions, on the second day of starvation. After 3 days of starvation, cells were stimulated with the indicated DHT concentrations and analyzed at the indicated time points. DHT and bicalutamide were purchased from Sigma.−1 mouse) dissolved in Miglyol 812, for three consecutive days. Finally, mice were killed, prostates were harvested, and RNA was extracted with the peqGOLD Total RNA Kit  according to the manufacturer's protocol. In vivo experiments complied with institutional guidelines for animal experimentation; ethical approval was obtained from the Federal Ministry for Science and Research, Vienna, Austria (BMWFW‐66.009/0332‐WF/V/3b/2015).Male C57Bl6/J mice aged 6–8 weeks were purchased from Charles River . At 8–10 weeks mice were either castrated or sham‐operated. Two weeks after surgery, mice were injected subcutaneously with Miglyol 812 (control) or with DHT . Serial deletions of the p278‐TF promoter were created with specific primers, as summarized in Table XhoI and BamHI digestion. The EGR1 Nanoluc reporter was created by digesting an EGR1 luciferase reporter  with AccI and HindIII. The released fragment was subsequently cloned into the pNL1.1 Nanoluc Reporter vector (Promega). Plasmids were checked by sequencing before use.p2106‐TF and p278‐TF fragments were derived from previously published plasmids m KH2PO4, 0.1% Triton X‐100). Nanoluc luminescence was measured as recommended by the manufacturer (Promega). Firefly luminescence and β‐galactosidase were measured as previously described Cells were transiently transfected with the TF gene promoter or NF‐κB and EGR1 reporter constructs, and a vector constitutively expressing β‐galactosidase for normalization purposes (driven by a ubiquitin promoter: PUB6/V5‐His/LacZ from Thermo Fisher Scientific). Cells were lysed in passive lysis buffer  was used as a housekeeping gene. Mean PCR efficiency was calculated with linreg−ΔΔCT values were used for calculation of the Pearson r coefficient (relative to TF expression), and statistical analysis was performed with graphpad prism 7.0 software.qPCR was performed with SYBR Green reagents (Thermo Fisher Scientific) as previously described The ABCD assay was performed as previously described m sodium citrate buffer (pH 6). HRP was developed with a Vectorlabs 3,3′‐diaminobenzidine peroxidase (HRP) Substrate Kit according to the manufacturer's protocol. Slides were counterstained with hematoxylin. The antibodies used for immunohistochemistry were anti‐TF (Abcam: AB151748) and anti‐EGR1 .Western blotting was performed according to standard procedures. The antibodies used were: anti‐p65 , anti‐p50 , anti‐β‐tubulin (Santa Cruz: sc‐9104), anti‐AR , anti‐TF , anti‐IκBα (Santa Cruz: sc‐371), anti‐c‐Rel , anti‐EGR1 (Santa Cruz: sc‐110), anti‐SP1 , and anti‐GAPDH . Immunohistochemistry was performed with a Vectastain Elite ABC horseradish peroxidase (HRP) Kit  according to the manufacturer's protocol. Antigen retrieval was performed by boiling slides for 20 min in 10 mTF activity was determined essentially as previously described m) for 48 h, harvested by scraping, and finally fixed in 1% paraformaldehyde. To determine the total cellular TF protein content in LNCaPs, cells were permeabilized with 0.1% Triton X‐100 in phosphate‐buffered saline (PBS) for 15 min, and labeled with anti‐CD142–fluorescein isothiocyanate (anti‐TF) . Mean fluorescence intensity (MFI) was analyzed with a BD Accurri C6 flow cytometer and BD Accuri C6 Samples software . MyC‐CaP cells were stimulated with different concentrations of DHT (1–100 nm) for 48 h, and detached with Versene solution at 4 °C (0.5 mm EDTA in PBS). To determine the surface TF protein content, MyC‐CaP cells were labeled with anti‐TF–phycoerythrin . Live cells were separated by staining with SYTOX AADvanced Dead Cell stain (Thermo Fisher Scientific). MFI was analyzed with a Cytoflex S cytometer and Cytexpert software 2.0 .LNCaP cells were stimulated with different concentrations of DHT  Overall, we believe, that our findings are especially important in the context of ADT, as it was previously shown that the VTE rate is increased in androgen‐deprived prostate cancer patients Taken together, our data indicate a novel, crucial role for AR in reducing TF expression, which could be important for increased TF expression and TF‐positive MV release in androgen‐deprived prostate cancer patients, and contribute to elevated VTE rates in prostate cancer patients.B. Hoesel conceptualized and coordinated the project, and designed experiments. B. Hoesel, A. Assinger, N. Mackman, and J. Schmid interpreted data and wrote the manuscript. B. Hoesel, M. Mussbacher, B. Dikorman, M. Salzmann, A. Assinger, B. Moser, and H. Paar performed experiments. L. Hell, J. Thaler, and U. Resch provided research tools. J. Basílio performed bioinformatics. All authors approved the final version of the manuscript.The authors state that they have no conflict of interest.Table S1. Primer sequences used in this study.Fig. S1. Gene set enrichment analysis of a published microarray dataset (GSE21032). TF, EGR1 and IκBα show an AR‐repressed gene signature.Click here for additional data file.


We decided to perform computer-aided analysis of the anterior cerebral artery (ACA) to check for a potential correlation with anterior communicating artery (ACoA) aneurysm presence and growth.We retrospectively analyzed the ACA anatomy of 121 patients with ACoA aneurysms along with 121 age, risk factors, and vessel side-matched control patients without an ACoA aneurysm. We obtained their medical history and digital subtraction angiography (DSA) data from their medical records. For each patient’s DSA, we extracted curve representing the course of their ACA and calculated its relative length (RL), sum of angle metrics (SOAM), triangular index (TI), product of angle distance (PAD), and inflection count metrics (ICM).p < 0.01), SOAM , PAD , and TI . In multivariate logistic regression analysis, after adjustment for possible confounders, SOAM  and TI  remained independently associated with higher risk of ACoA aneurysm. Additionally, we found significant negative correlations between TI and aneurysm dome size .Patients with ACoA aneurysm had significantly higher RL (0.64 ± 0.23 vs. 0.56 ± 0.22; Increased tortuosity of ACA might increase the risk of ACoA aneurysm development and decrease the risk of aneurysm growth.• Anterior cerebral artery’s sum of angle metrics is associated with hypertension as well as with history of ischemic stroke and myocardial infarction.• Increased tortuosity of anterior cerebral artery might be associated with anterior communicating artery aneurysm development.• Tortuosity of anterior cerebral artery is negatively correlated with anterior communicating artery aneurysm dome size. Vessel tortuosity has been found in a number of organ systems , is assoWe hypothesize that ACA tortuosity might be related to the risk of ACoA aneurysm formation and therefore we performed computer-aided analysis of ACA tortuosity and its relation with the presence, type, and propensity for progression of ACoA aneurysms.We retrospectively analyzed data of patients with intracranial aneurysm, confirmed by digital subtraction angiography (DSA), hospitalized between January 2013 and November 2017, at the Department of Neurosurgery, Jagiellonian University, University Hospital, Kraków, Poland. Patients who underwent clipping or coiling of the aneurysm were excluded from our study. We selected 121 patients with ACoA aneurysms. The control group consisted of 121 patients without an intracranial aneurysm or with aneurysm located on different arteries than ACoA, ACA, or internal carotid artery (ICA). We aimed to match the control group for age (± 2 years), side of ACA, and risk factors including hypertension, diabetes mellitus, and smoking. From patients’ medical records, we obtained their medical history, including previous and current diseases and medications. Patients with multiple aneurysms and connective tissue disorders were excluded from our study. The study protocol was approved by the Ethics Committee of the Jagiellonian University and written consent was obtained from each patient included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. The data that support the findings of this study are available from the corresponding author upon reasonable request.To perform all image transformations and factor calculations, we used original software written by the first author of this study in C# 7.0 using Microsoft Visual Studio Community 2017 (Microsoft Corporation) licensed for non-commercial use together with Emgu CV image processing library.To detect ACA course, we performed a series of image transformation on the lateral projection of each patient’s DSA. First, the bone structures were subtracted and we performed gamma correction to increase visibility of blood vessels. Then, we applied multiscale vessel enhancement filter  to detecnth step, semi-ellipse shape-searching region was defined with center On, major axis length an, minor and axis length bn, and a direction vector On was defined as the midpoint between previously found edge points, dn is the distance between two previously selected points and θn is the angle between nth step was treated as vessel crossing or branching, and points located in that step were not taken into consideration. Curve-connecting midpoints between each pair of edge points, representing the ACA course, were used for further analysis .t test for normally distributed variables and the Mann-Whitney U test for non-normally distributed variables. We used the χ2 test for the dichotomized variables. To assess the correlation between continuous variables, we used Pearson’s or Spearman’s correlation tests, for normally and non-normally distributed variables, respectively. We express continuous variables as mean ± standard deviation. To find factors independently associated with the presence of the ACoA aneurysm, we employed logistic regression analysis, with and without adjustment for possible confounders. All significance tests are two-tailed and the p value of < 0.05 has been considered significant.We used the Shapiro-Wilk test to assess normality. For comparisons of continuous variables, we used the The mean age of 242  patients was 59.2 ± 12.6 years. The indices of ACA anatomy were as follows: the mean diameter of A1 segment was 1.77 ± 0.44 mm, mean diameter of A2 segment was 1.47 ± 0.43 mm, mean diameter of A3 segment was 1.28 ± 0.38 mm, and mean length of A1–A3 segments was 10.27 ± 2.17 mm. The average diameter of ICA C7 segment was 3.47 ± 0.78 mm. For A1–A3 ACA segments, mean RL was 0.60 ± 0.23, mean SOAM was 0.23 ± 0.18, mean PAD was 0.11 ± 0.12, mean TI was 0.50 ± 0.16, and mean ICM was 0.09 ± 0.12. Among patients with ACoA aneurysm, average dome size was 4.95 ± 2.82 mm. Among patients in the control group, 72 (59.02%) had intracranial aneurysm. The most common location of these aneurysms was the middle cerebral artery (54.16%), then the basilar artery (29.17%), posterior communicating artery (13.88%), and vertebral artery (2.78%).p = 0.01), as was PAD  and TI . Additionally, patients with hypertension had significantly higher SOAM . We found no significant differences in tortuosity indices according to the presence or absence of diabetes mellitus, hypercholesterolemia, or active smoking. However, patients with a history of myocardial infarction had significantly higher SOAM . Similarly, SOAM was significantly higher among patients with a history of ischemic stroke  , less often took angiotensin-converting enzyme inhibitors (ACEI) , and more often took anticoagulants . In terms of artery sizes, patients with ACoA aneurysm had significantly longer A1–A3 ACA segments  and significantly thinner A3 segment . They also had significantly higher RL , SOAM , PAD , and TI . We found no significant difference in terms of ICM between patients with and without ACoA aneurysm   (Table p < 0.01) and TI  remained independently associated with higher risk of ACoA aneurysm and female gender  remained independently associated with lower risk of ACoA aneurysm.Patients with ACoA aneurysm more often were male  and arterial hypertension (SOAM).Our study showed a significant correlation between higher tortuosity descriptors and certain risk factors of aneurysm development. Coexistence of arterial hypertension and higher vascular tortuosity had been widely described in relation to retinal  and coroWe also found that patients with ACoA aneurysm had higher SOAM, PAD, and TI. They also had higher RL. These findings may suggest that local smaller angles in ACA course could impact more on aneurysm development than deviations from a straight line. It is important to note that the natural course of the ACA significantly departs from straight line. Therefore, the RL may not be a suitable tortuosity index of this artery. Higher PAD and TI values for ACoA aneurysm patients may imply that global tortuosity is also higher in arteries of those patients. To the best of our knowledge, only one study analyzed the impact of ACA tortuosity on ACoA aneurysm development; however, the results were inconclusive . SeveralAn explanation for the association of aneurysm development with higher tortuosity is most likely due to the impairment of the arterial wall. Hemodynamic studies, performed on coronary arteries, showed that higher tortuosity is associated with lower wall shear stress (WSS), prolonged relative residence time (RRT), and disturbed blood flow . Higher An interesting finding of this and our previous study, concerning MCA , is the In our previous study, we also found that lower tortuosity of ACA is related to higher risk of ACoA aneurysm rupture . It is cHigher global tortuosity and smaller local angles may contribute to intracerebral aneurysm development, most likely due to the higher risk of arterial wall impairment and further remodeling. However, increased tortuosity, following a decrease in blood flow and wall shear stress, could be a protective factor against aneurysm growth.