A range of tonicities (isotonic to hypotonic) was present at day 3 in the PEG‐treated samples. However, during culture, the tonicity range narrowed as GAGs leached from the tissue. TNF‐α and IL‐1β were below detection limits in all conditions, while mid‐ and downstream inflammatory cytokines were detected. This may suggest that the extracellular environment directly affects NP cells instead of inducing a classical inflammatory cascade. Furthermore, IL‐8 increased in swelling restricted samples, while IL‐6 and PGE2 were elevated in free swelling controls. These findings may suggest the involvement of different mechanisms in disc degeneration with intact AF compared to herniation, and encourage further investigation. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop ResInflammatory cytokines play an important role in intervertebral disc degeneration. Although largely produced by immune cells, nucleus pulposus (NP) cells can also secrete them under various conditions, for example, under free swelling. Thus, tissue hypotonicity may be an inflammatory trigger for NP cells. The aim of this study was to investigate whether decreased tonicity under restricted swelling conditions (as occurring in early disc degeneration) could initiate an inflammatory cascade that mediates further degeneration. Healthy bovine NP tissue was balanced against different PEG concentrations (0–30%) to obtain various tissue tonicities. Samples were then placed in an artificial annulus (fixed volume) and were cultured for 3, 7, or 21 days, with free swelling NP as control. Tissue content  was analyzed, and both the tissue and medium were screened for tumor necrosis factor alpha (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin‐E Low back pain is the most common cause of chronic pain in the aging western world.The healthy intervertebral disc consists of a gel‐like core, the nucleus pulposus (NP), which is circumferentially surrounded by several highly organized fibrous layers which together form the annulus fibrosus (AF). The NP and inner AF are cranially and caudally separated from the vertebrae by the porous hyaline cartilage endplates, through which nutrients diffuse. Several factors are of influence for disc degeneration, for example, genetic inheritance, impaired nutrient supply, and mechanical loading. However, the exact cause and developmental path are still unclear.2 (PGE2), are also detected in NP tissue without herniation,Although inflammation is most profound after herniation, pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and prostaglandin‐EThe pro‐inflammatory cytokines, present in extruded NP tissue, are mainly attributed to the body's immune response.l‐ascorbic acid 2‐phosphate, A8960, Sigma), and 1% penicillin/streptomycin  under physiological conditions . Medium was changed twice a week, collected, and stored at −80°C until further use. At days 3, 7, and 21, NP samples were removed from the artificial annulus, weighed, snap frozen, and stored at −80°C until further use.Bovine caudal discs  were obtained from the local abattoir in accordance with local regulations. The discs were transversally opened at the cartilage endplate/NP interface, and NPs were harvested from the central part of the disc with a biopsy punch . From each tail, one NP was weighed and snap frozen directly after harvesting as base‐line measurement (day 0). The other NPs were weighed and equally distributed (disc level per donor) over the culture groups (Table S1). Next, each NP was placed in dialysis tubing  and pre‐shrunk in 30%, 20%, 10%, or 0% w/v polyethylene glycol  in PBS for 100 min, depending on the assigned group (Tables S1 and S2). Thereafter, the dialysis tubing was removed, NPs were weighed, wrapped tightly in a 0.2 µm pore size regenerated cellulose membrane , and placed in an artificial annulus consisting of custom jackets  knitted from Dyneema Purity fibers .l‐cystein, 5 mM ethylenediaminetetra‐acetic acid, and 140 µg/ml papain, all from Sigma) at 60°C. The DNA content of the digested samples was measured with the Qubit Fluorometric Quantitation method . The glycosaminoglycan (GAG) content, as a measure for the amount of proteoglycans, was determined with a DMMB assayTo assess the biochemical content in the tissue, one‐fourth of each NP sample was weighed (wet weight), lyophilized  overnight and weighed again (dry weight). The water content was calculated by dividing the difference between wet and dry weight by the wet weight. Subsequently, the samples were digested overnight in papain digestion buffer , as described by van Dijk et al.2  were measured in the medium by ELISA. The concentrations were normalized to the initial sample wet weights to obtain the overall tissue response. If possible, standard curves were prepared in culture medium to account for medium effects. If this was not possible, the cytokine concentration in the medium was determined as control on the plate.Furthermore, the concentrations of TNF‐α, IL‐1β, IL‐6, IL‐8, and PGEThe nitric oxide (NO) concentration in the medium (N = Table S1) was measured as described by Miranda et al.p < 0.05.Statistical analysis was performed using R‐project software (version 3.0.2).Using the PEG system for pre‐shrinking of the samples, the wet weight of the samples could be initially (day 0) adjusted to a range from 59 ± 7% to 171 ± 26% of the initial wet weight  increased to almost 500%, while a decrease of approximately 40% was measured in the 0% PEG constrained group (±310 mOsm). Furthermore, a layer of densely packed cells was observed on the periphery of the free swelling samples (data not shown).2, and NO were measured in both the tissue and medium of all cultured samples. However, in the tissue samples, all markers were below the detection limit. In addition, IL‐1β and TNF‐α concentrations were also below the detection limit  in the medium of all culture conditions at all time points (positive control: IL‐1β stimulated NP tissue). IL‐6 levels were only below the detection limit in the medium of the constrained samples (0–30% PEG groups), while an increasing concentration was measured in the free swelling samples between days 0 and 21  disc degeneration and herniation, were absent.To evaluate if a decrease in tonicity of constrained NP tissue triggers an inflammatory cascade, healthy bovine NP tissues were balanced against different PEG concentrations to obtain a range of tonicities (isotonic to hypotonic). During culture, a short‐term but tonicity‐independent inflammatory response of the NP cells was observed. Surprisingly, only mid‐stream and downstream inflammatory cytokines, for example, IL‐8, PGE2 and NO cytokines. IL‐8 has been identified as a potent chemoattractant for neutrophils and lymphocytes and has been associated with the development of radicular pain.2, similar as reported for herniated tissues.Furthermore, differences in inflammatory reactions were observed between constrained hypotonic (0% PEG) samples and free swelling samples, even though they had similar tonicities. Culture of constrained NP tissues, including the 0% PEG group, resulted in an increase of IL‐8 production and a temporal elevation of PGEIn addition, differences at tissue level were observed between constrained hypotonic (0% PEG) samples and free swelling samples. The DNA content of constrained samples decreased to 50% of the initial content, while it increased 600% in the free swelling samples. Indeed, an increase in spindle shaped cells, which formed several cell layers around the tissue periphery, was observed in the free swelling samples, explaining the increase in DNA content. These spindle‐shaped cells were previously identified in vitro as dedifferentiated NP cellsA range of tonicities was obtained at day 3 between the different PEG‐treated NP tissues (320–430 mOsm). In human NP tissue the tonicity reduces during disc degeneration with ∼25% between grade 2 and 3 of the Thompson scale, and ∼35% between grades 2 and 4 .Although a tonicity range was obtained at day 3, differences between groups reduced during culture, and thus similar tonicities were observed in all constrained samples at day 21 (310–350 mOsm). Contrarily, differences in the water content due to the PEG procedure remained stable during culture as would be expected by the limited swelling from the artificial annulus. This indicates that the time‐related drop in tonicity, calculated from the amount of proteoglycans per wet weight, is caused by the loss of proteoglycans. Indeed, the GAG content reduced during culture in all sample groups, and relatively high GAG concentrations were measured in the culture medium at day 3. This observation is in contrast to our previous study where the proteoglycan content in the (isotonic) 30% PEG‐treated group could be maintained for 42 days.2 and NO was measured at day 3 when the tonicity range was still present, which decreased again during the remaining culture when the tonicity range was lost. Prolonged elevation of NO has been associated with apoptosis,Furthermore, an increase of PGEIn addition to apoptosis, elevated NO levels are associated with increased matrix breakdown and reduced proteoglycan synthesis.2, NO) were produced by the NP cells, implying that the extracellular environment directly affected NP‐cells or that other upstream mechanisms are involved. This observation challenges the hypothesis that NP cells generate a classical inflammatory cascade within the degenerating disc. Overall, these findings encourage further evaluation of the role of NP cells in the inflammation pathways observed during disc disease. Gaining better understanding of the inflammatory mechanisms will help to identify novel targets for the treatments of disc degeneration.A short‐term inflammatory response was observed in constrained NPs, with only limited differences in short‐term exposure to a range of isotonic to hypotonic tissue conditions. However, different inflammatory markers were produced by NP cells in constrained and unconstrained tissue with the same hypotonicity. These results suggest that different processes may be activated in NP tissue in degenerating discs (constrained NP) compared to herniated discs (unconstrained NP). Furthermore, upstream inflammatory cytokines identified as key players in disc degeneration  were undetectable in all conditions, while mid‐ and downstream cytokines (IL‐6, IL‐8, PGEV.M.: writing of manuscript, data interpretation, generated figures. I.A.: study design, experimental work, data interpretation, writing of manuscript. B.vD.: study design, data interpretation, revising manuscript. K.W.K.: study design, critically reviewed manuscript. K.I.: study design, data interpretation, critically reviewed manuscript. All authors have read and approved the final submitted manuscript.Additional supporting information may be found online in the Supporting Information section at the end of the article.Table S1. Number of independent samples per group and time point for both the tissue and culture medium.Table S2. Sample groups and wet weight change per group after being balanced against different PEG concentrations.Click here for additional data file.


Three-dimensional ultrasound (3DUS) is an attractive option in image-guided radiotherapy (IGRT) for prostate cancer (PCa) patients. However, the potential factors influencing the accuracy of 3DUS in comparison with cone-beam CT (CBCT) in IGRT for PCa patients haven’t been clearly identified.The differences between US/US and CBCT/CT registrations were analyzed over 586 and 580 sessions for 24 and 25 PCa patients treated with or without pelvic lymph node irradiation, respectively. The clinical factors that may influence registration differences were also evaluated.The average discrepancies between US/US and CBCT/CT registrations were − 0.28 ± 5.28 mm, − 0.16 ± 3.48 mm, and − 0.47 ± 4.31 mm in the superior-inferior (SI), left-right (LR), and anterior-posterior (AP) directions, respectively. The discrepancies were respectively less than 5 mm longitudinally, laterally, and vertically in 64.4 and 70.1%, 84.9 and 89.2%, and 75.9 and 79.1% of the patients treated with or without pelvic lymph node irradiation, respectively. The registration differences were significantly smaller at least in one direction in patients younger than 70 years, without pelvic lymph node irradiation, guided by transperineal ultrasonography and had a bladder volume smaller than 300 mL.Age, irradiated regions, 3DUS modality, and bladder volume are important factors that may influence the differences between US/US and CBCT/CT registrations. 3DUS guidance is more feasible for younger PCa patients with a better control of bladder volume during the treatment and those who did not undergo pelvic lymph node irradiation.The online version of this article (10.1186/s13014-019-1217-0) contains supplementary material, which is available to authorized users. Image-guided radiotherapy (IGRT) is an essential prerequisite for accurate dose delivery in prostate cancer (PCa) radiotherapy –4. AlthoThe Clarity System  is one of the latest-generation US-based guidance systems that have been shown to be feasible for IGRT in PCa radiotherapy –14. HoweTo date, no study has evaluated the feasibility of the 3DUS system for PCa patients treated with pelvic lymph node irradiation. However, pelvic lymph node irradiation is usually indicated for locally advanced PCa patients in China. Therefore, we conducted this study to explore the feasibility of using the Clarity 3DUS system for PCa patients treated with or without pelvic lymph node irradiation and to identify the relative influencing factors.This retrospective study evaluated the setup verification for 49 patients who received external beam radiation therapy for localized prostate cancer from June 2015 to February 2017 at Sun Yat-sen University Cancer Center. Thirty patients underwent definitive radiotherapy with a total dose of 67.5 Gy over 25 fractions, and the other 19 patients received adjuvant radiotherapy after prostatectomy with a total dose of 64–72 Gy over 32–36 fractions. Each patient was treated with a 6-MV linear accelerator  with a volumetric modulated arc therapy (VMAT) plan. Both 3DUS and CBCT scans were repeated before each treatment if possible after obtaining patient consent. This study was approved by the ethics committee of the Cancer Center of Sun Yat-sen University. Written informed consent was obtained from all the patients before treatment.ref). For TAUS scanning, the US probe was manually placed 5–10 cm supra-pubic on the abdomen with a moderate pressure for a good quality of US images. Then the US probe is swept from superior to inferior, without translatory movement of the probe, to scan the prostate and bladder from retropubic to the top of bladder. For TPUS scanning, the transperineal US probe was placed on the perineum with a moderate pressure for good image quality, which allowed complete visualization of the penile bulb, prostate, and bladder. The US images were acquired using transabdominal ultrasonography (TAUS) for 30 patients undergoing definitive radiotherapy and using transperineal ultrasonography (TPUS) for the other 19 patients receiving adjuvant radiotherapy after prostatectomy.The workflow of Clarity was recommended by the manufacturer and was shown in Additional file ref image. For patients undergoing definitive radiotherapy, the RPV was the entire prostate. For those undergoing post-prostatectomy radiotherapy, the RPV corresponded to the bladder neck since it is included in the CTV according to the European Organization for Research on Treatment of Cancer (EORTC) guideline , and odds ratios (ORs) and 95% confidence intervals were reported.All statistical analyses were performed using SPSS 20.0 , and Forty-nine prostate cancer patients who underwent 1340 treatment sessions were included in this study. CBCT examinations were successfully performed in a total of 1331 sessions (99.33%) and 3DUS examinations were successfully performed in 1175 sessions (87.69%). Insufficient bladder filling was the most frequent obstacle for successful 3DUS. A total of 1166 paired US and CBCT translational shifts were calculated. All the following analyses are based on data from these 1166 paired shifts, and the accuracy evaluation of 3DUS was presented on the basis of the values provided by the soft-tissue match in CBCT.Setup errors in target localization between CBCT and the simulation CT were − 0.77 ± 3.98 mm, 0.05 ± 3.43 mm, and − 0.14 ± 3.33 mm in the SI, LR, and AP directions, respectively. For US imaging, setup errors were − 0.48 ± 6.09 mm, 0.23 ± 4.17 mm, and 0.40 ± 5.19 mm in the SI, LR, and AP directions, respectively  or LR (P = 0.124) directions but showed significant differences in the AP direction (P < 0.001). Shift agreements for a range of ±5 mm were determined, and 67.6, 87.3, and 77.7% of the discrepancies were less than 5 mm longitudinally, laterally, and vertically, respectively. The upper and lower LOA values were 10.07 mm/− 10.63 mm, 6.66 mm/− 6.98 mm, and 7.98 mm/− 8.92 mm in the SI, LR, and AP directions, respectively  and in none of the directions in group B  or not (group B). As shown in Fig. P = 0.899; LR, P = 0.151; AP, P = 0.103), but were significantly different in all directions in the TAUS group . However, the rectum volume showed no significant influence on the discrepancies between 3DUS and CBCT in any direction. Treatment without pelvic lymph node irradiation, bladder volume < 300 mL, and TPUS were significant influencing factors for the discrepancies between 3DUS and CBCT in the SI  and LR directions , in the LR (P = 0.004) and AP directions (P = 0.031), and in the LR direction , respectively  . We wereWe further compared the 3DUS and CBCT shifts in patients who underwent pelvic lymph node irradiation and those who did not, and found that the shift agreement was higher in all directions for those who did not undergo pelvic lymph node irradiation. The mean discrepancies were significantly different in all directions for patients who underwent pelvic lymph node irradiation but in none of the three directions for those who did not undergo pelvic lymph node irradiation. These data suggest that the irradiation area is an important factor influencing the accuracy of 3DUS with reference to CBCT for radiotherapy in prostate cancer.In contrast of the large registration discrepancies between TAUS and CBCT mentioned above, Fargier-Voiron et al. reported a better shift agreement in comparisons of TPUS and CBCT, with values above 90% in all directions, except the AP direction (76.6%) for the prostate and the SI direction (85%) for post-prostatectomy cases . In accoInterestingly, both age and bladder volume were identified as important factors that affect the accuracy of 3DUS with reference to CBCT. We suppose that the actual underlying factor is the repeatability of the bladder volume during the course of treatment. Although all patients were asked to hold a filled bladder in order to reduce the dose exposure to their bladder and intestine, patients younger than 70 years usually had better control of the bladder volume than the older patients. On the other hand, a bladder volume of less than 300 mL is easier for the PCa patients to repeat. Moreover, an excessively filled bladder may increase the urge to urinate and the risk of movement during the treatment. Taken together, our findings suggested that 3DUS is more feasible for PCa patients younger than 70 years with a filled bladder of no more than 300 mL.Our study has some limitations: first, the impact of the probe pressure was not analyzed in our study since we did have any device to monitor the probe pressure during each treatment; second, since all US images for patients undergoing definitive radiotherapy were acquired by TAUS, and all the other US images for patients receiving adjuvant radiotherapy after prostatectomy were acquired by TPUS, we only considered the US modality but not the radiation purpose (definitive or adjuvant) in the analysis. Further studies that include more potential impact factors and larger patient populations are still needed to confirm our results.3DUS guidance is not safely interchangeable with CBCT for pre-treatment repositioning in all PCa patients. Age, irradiated regions, 3DUS modality, and bladder volume are important factors that may influence the accuracy of 3DUS with reference to CBCT in image-guided radiotherapy for prostate cancer. 3DUS guidance is more feasible for younger PCa patients with a better control of bladder volume during the treatments and those who are not receiving pelvic lymph node irradiation.Additional file 1:Figure S1. The workflow of Clarity System recommended by the manufacturer. (TIF 703 kb)Additional file 2:Figure S2. Daily quality control. Daily quality control data of the Clarity system from June 2015 to February 2017 at Sun Yat-sen University Cancer Center were shown. The Clarity™ calibration phantom was used to measure the discordance between Clarity and CBCT. (TIF 123 kb)Additional file 3:Table S1. Comparison of setup errors in 3DUS versus CBCT for all patients in all three directions. (DOCX 18 kb)Additional file 4:Table S2. Inter-operator variability of the registration of CT/CBCT and US/US images. (DOCX 15 kb)


Objectives. To evaluate PS expression, circulating MPs, and the corresponding PCA in RVO patients. Twenty-five RVO patients were compared with 25 controls. PS-positive cells were detected by flow cytometry. Cell-specific MPs were measured by lactadherin for PS and relevant CD antibody. We explored PCA with coagulation time, purified coagulation complex assays, and fibrin production assays. In RVO, MPs from platelets, erythrocytes, leukocyte, and endothelial cells were increased and the exposure of PS was elevated significantly when compared with controls. In addition, we showed that circulating MPs in RVO patients were mostly derived from platelets, representing about 60–70% of all MPs, followed by erythrocytes and leukocytes. Moreover, PS exposure, ECs, and MPs in RVO lead to shortened clotting time with upregulation of FXa and thrombin formation obviously. Importantly, ECs treated with RVO serum which bounded FVa and FXa explicitly suggested the damage of retinal vein endothelial cells. Furthermore, lactadherin can inhibit the combination between PS and coagulation factors by approximately 70% and then exert an anticoagulant effect. In summary, circulating MPs and exposed PS from different cells may contribute to the increased PCA in patients with RVO. Lactadherin can be used for PS detection and an anticoagulant agent.The pathogenesis of hypercoagulability in retinal vein occlusion (RVO) is largely unknown. Whether the exposure of phosphatidylserine (PS) and microparticle (MPs) release will affect procoagulant activity (PCA) in RVO needs to be investigated.  Retinal vein occlusion (RVO) is a unilateral blinding disease including branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). It was confirmed and demonstrated that people affected by RVO around the world were second to diabetic retinopathy . The exiμm in diameter and have different cellular origins like blood cells, endothelial cells, and other tissues [Phosphatidylserine (PS) is an anionic phospholipid, generally distributed in the cell membrane. When the cells are activated or upon apoptosis, PS will externalize to the outer membrane and combine with FV and FVIII to enhance the procoagulant activity (PCA) . During  tissues . Overpro tissues –14. Howe+ cells and MPs in CRVO.Our aim was to explore the externalization of PS and MPs in RVO and further analyze their PCA. We investigated the MP release and PS exposure in RVO patients which may be important for the thromboembolic events of RVO. Lactadherin, a new sensitive probe and anticoagulant, can compete with PS for binding clotting factors V and VIII and was compared with annexin V in RVO patients . TherefoThis present study was permitted by each patient and agreed with the ethics committee of Harbin Medical University on the basis of the Helsinki Declaration. Enrollees were 25 patients newly diagnosed with RVO (10 CRVO and 15 BRVO) who were admitted between July 2015 and December 2016, compared with 25 healthy controls who were from a physical examination center, similar in age, sex, and associated pathologies. Except for the medical record, the ophthalmologist conducted a retinal angiography to collect ophthalmological data. RVO was diagnosed when the retinal area appeared superficial and there is a deep intraretinal flame-shaped haemorrhage. We should exclude the RVO patients who had accepted intravitreal ranibizumab, vitrectomy, or retinal laser treatment before. Some other factors affecting blood coagulation (exclude) are as follows: diabetes, hyperhomocysteinemia, antiphospholipid antibody syndrome, glaucoma, and so on .g for 2 min to gain platelet-free plasma (PFP). PFP was stored in liquid nitrogen immediately. Gradient centrifugation was used to get leukocytes. PFP was centrifuged for 45 min at 20,000 ×g to get MPs. Then the supernatant (MP-depleted plasma (MDP)) was washed with 25 μl MPs.Tubes filled with blood samples were centrifuged for the isolation of platelet poor plasma (PPP). PPP was carefully taken from the top of the tube, without disturbing the buffy coat, and centrifuged at 13000μl) were stored in 35 μl Tyrode's buffer and incubated for 10 min at room temperature in the dark Then it was analyzed by flow cytometry (FACS Canto II). To detect PS-positive platelets, erythrocytes, neutrophils, and endothelial cells, samples  were incubated with Alexa 488-conjugated lactadherin+ CD41a (5 μl), Alexa 488-conjugated lactadherin+ CD235a (5 μl), Alexa 647-conjugated lactadherin+ CD45 (5 μl), or Alexa 488-conjugated lactadherin+ CD31+CD41a− (5 μl). Flow cytometry (FACS Canto II) was used to test the exposure of PS on PlTs, RBCs, WBCs, and ECs binding with lactadherin. Platelet MPs (PMPs), RBC MPs (RMPs), neutrophil MPs (NMPs), and endothelial cell-derived MPs (EMPs) were defined by lactadherin+ CD41a+, lactadherin+ CD235a+, lactadherin+ CD66b+, and CD41a+/CD31−, respectively, and other cell origins by their specific monoclonal antibodies.MPs  were dealt with media containing 20% of pool serum from 25 different RVO patients or donors at room temperature for 24 h. The PS exposure was defined by a flow cytometer. Each test was done in duplicate.Human umbilical vein endothelial cells (HUVECs) were cultured with endothelial cell medium  at 37°C in a 5% COμl platelets (1 × 107/ml), erythrocytes (1 × 108/ml), leukocytes (1 × 106/ml), endothelium (1 × 106/ml), and MP suspension were incubated with 100 μl MP-free human plasma for 180 s. One hundred μl of 25 mM CaCl2 was added. For inhibition assay, 50 μl lactadherin or anti-TF antibody was incubated with blood cells and MP suspension as before for 10 min. Clotting time was recorded.The coagulation time of platelets, RBCs, WBCs, ECs, and MPs was detected in a STart4 coagulometer (Diagnostica Stago). One hundred μl platelets (2 × 104/ml), erythrocytes (2 × 105/ml), leukocytes (2 × 104/ml), endothelium (2 × 104/ml), and MP suspensions with 1 nM factor IXa, 130 nM factor X , 5 nM factor VIII, 0.8 nM thrombin , and 5 mM Ca2+ in factor Xase buffer at room temperature. The reaction deals with the addition of EDTA. Subsequently, chromogenic substrates S-2765  were added to each reaction system, and factor Xa product was determined immediately at 11 s interval for 15 min. For the extrinsic FXa formation assay, 2 × 104 platelets were incubated with 130 nM FX, 1 nM FVIIa, and 5 mM Ca2+ for 5 min. The reaction was quenched by EDTA, and the amount of FXa formation was detected as described above. For prothrombinase formation assay, the samples were incubated with 1 nM factor Va, 1 μM prothrombin , 0.05 nM factor Xa , and 5 mM Ca2+ at room temperature. Stop buffer EDTA and then 10 μl Chromogenix S-2238  were added into each microplate to evaluate the production of thrombin. Cells and MPs were dealt with varying concentrations of lactadherin (0–128 nM) or anti-TF antibody for 10 min in Tyrode's buffer. The mixture was dealt with the clotting factors. Then the production of FXase or prothrombinase was detected.Intrinsic factor Xase formation assay begins with the incubation of 10 FVa/Xa binding was observed with confocal microscopy; ECs were costained with factor Va-fluorescein-maleimide and factor Xa-EGRck-biotin (complexed to Alexa 647-steptavidin). Fibrin formation was quantified by turbidity in a SpectraMax 340PC plate reader . ECs culCorrelations between MPs and APTT and PT and FIB were done using Pearson's correlation coefficients. The Spearman rank correlation was used to detect discrete variables.t-test. P < 0.05 was considered statistically significant.SPSS14.0 statistical software  was used for the analysis of each observation time point and each group. Data expressed as the mean ± standard deviation (SD) were made. Pearson's correlation coefficient was used. The comparison between different groups was analyzed by one-way ANOVA. The comparison between the two groups was analyzed using Student's Clinical characteristics and blood laboratory results of retinal vessel occlusion patients and controls are presented in + MPs between the RVO group (1547 ± 83/μl) and the controls . Flow cytometry analysis result indicated that exposure of PS on platelets, red blood cells, leukocytes, and endothelial cells was significantly increased in retinal vein occlusion groups than controls . The levels of MPs in RVO were detected because of PS exposure. There was a significant number of lactadherin+ MPs between the RVO group and the control group (P < 0.05). Lactadherin was also used to detect PS exposure of erythrocytes, leukocytes, and endothelial cells in each study group by confocal microscopy . In inhibition assays, lactadherin (at 128 nM) blocked the production of the three procoagulant enzyme complexes by nearly 70%  were significantly related to FIB in patients with RVO , prothrombin time (PT), and fibrinogen (FIB) in healthy subjects and patients with RVO. Our results revealed that circulating MPs  phosphorylation on red blood cells from polycythemia vera (PV) patients may be crucial in these disorders , 23. In membrane , supportmembrane . Howevermembrane –28; thenECs also play an important role in maintaining optimal homoeostasis and blood fluidity within the intravascular space by balancing coagulation and fibrinolytic systems. Previous studies have found that the site-specific changes of vascular endothelial phenotype may be linked to the endothelium dysfunction in central or branch retinal vein occlusion and ECs are thought to be one of the key players in the pathogenesis of RVO , 30. NotIn summary, we illustrate the potential mechanisms of coagulation abnormalities in RVO that blood cells, endothelium, and circulating MPs participate in exuberant PCA via their PS exposure. More importantly, our results suggest the alterations in fibrin formation and deposition on endothelial cells in the pathogenesis of RVO. The differences in PS exposure and MP release cannot lead to cardiovascular risk factors because they were spread in the RVO and the control groups. The limitations of our study are the size of the population and the medical history of different lengths of time. PS inhibition assays demonstrate that the inhibition of PS might be a novel anticoagulant therapy strategy.


GATA4 in a Japanese family with atrial septal defect, including boys with apparently normal male sex development. The findings, together with the previous data, imply that GATA4 variants primarily cause congenital heart disease and rarely result in 46,XY disorder of sex development.We identified a heterozygous p.(R284H) variant of  They were born at term with normal birth sizes, and exhibited normal growth and mental development. In the yearly follow‐up observations, physical examinations showed no minor anomalies, and routine blood and urine laboratory tests including hepatic and renal function markers indicated normal findings. On the last examinations at 7, 5, and 3 years of age, respectively, they remained healthy without medication, and bone survey and visceral ultrasonographic studies revealed no abnormal findings. The parents and the ASD2‐negative III‐4 were also clinically normal.We performed whole‐exome sequencing (WES) with SureSelect Human All Exon V6 , using leukocyte genomic DNA of II‐1, II‐2, III‐2, III‐3, and III‐4. This study was approved by the Institutional Review Board Committee at Hamamatsu University School of Medicine, and performed after obtaining written informed consent. Captured libraries were sequenced by NextSeq 500  with 150‐bp paired‐end reads. Reads were aligned to the reference genome (Human GRCh37/hg19), using BWA‐MEM (version 0.7.12) with default parameters. Duplicated reads were removed by Picard (version 2.9.2), and local realignment and base quality recalibration were performed by GATK version 3.7. Variants were identified with the GATK HaplotypeCaller, and variants with minor allele frequencies of <0.005 in all the following public databases and in‐house database were selected as rare variants: whole genome and WES data for East Asian population in Genome Aggregation Database,GATA4 ; CRCh37, Chr8:11607687) as the most likely candidate variant for ASD2 in this family  and absent from the ASD2‐negative subjects (II‐1 and III‐4) Table . We nexty Figure . This vaGATA4 variant, we investigated the status of male sex development in two variant‐positive subjects (III‐1 and III‐2)  allegedly had TOF. Previous studies have also revealed variousGATA4 variant in this family, because previous studies have shown that GATA4 variants, though they are occasionally accompanied by extra‐cardiac features such as 46,XY DSDNo clinically recognizable extra‐cardiac feature was identified in the ASD2‐positive subjects in this family, although it might be possible that some extra‐cardiac feature(s) have remained undetected. This would primarily be consistent with theAMH promoter and enhances AMH expression synergically with NR5A1,GATA4 p.(R284H) variant may have primarily impaired AMH expression, with no clinically discernible deleterious effect on other Sertoli cell function including Sertoli‐germ cell interaction. Indeed, male subjects with the same p.(R284H) variant in the French family are fertile, although they have ASD2.GATA4 p.(R284H) variant on testicular function would remain at a subclinical level, if any. It should be pointed out, however, that GATA4 variants have been identified in five patients with 46,XY DSD.GATA4 variants lead to 46,XY DSD in rather exceptional subjects, depending on the residual activity of the GATA4 variants and the predisposing genetic and environmental factors of the variant‐positive subjects.Clinical and endocrine studies were performed for male sex development in two variant‐positive boys. In this regard, although well‐masculinized external genitalia and normal LH and testosterone productions indicate well‐preserved Leydig cell function, slightly increased FSH values and obviously low AMH values may suggest more or less compromised Sertoli cell function in the two boys.4GATA4 variant in a family with dominantly inherited ASD2 by WES. The results, together with the previous findings, imply that GATA4 variants primarily lead to congenital heart disease and rarely result in extra‐cardiac features including 46,XY DSD.We identified a The authors declare no conflict of interest.DS: performed whole‐exome sequencing and drafted the manuscript. SI, KS, and SH: performed the patient care and collected clinical information. MF: performed the whole‐exome sequencing in co‐operation with DS. HS: supervised the whole‐exome sequencing and performed the data mining. TO: coordinated the study and wrote the manuscript. Click here for additional data file.


Kawasaki disease (KD) patients who are unresponsive to intravenous immune globulin (IVIG) have a high occurrence of coronary artery lesions (CALs). The characteristics of left ventricular (LV) function alternation in IVIG‐resistant patients are not well‐described.Two‐dimensional speckle tracking echocardiography (STE) is a useful technique that can accurately detect myocardium subclinical dysfunction in resistant patients and may assist in differentiating patients with KD at a higher risk of IVIG resistance.A consecutive sample of 50 IVIG‐resistant patients , 50 IVIG‐responsive patients  and 50 normal subjects  were analyzed using STE, and receiver operating characteristic curve (ROC) analysis was utilized to determine the threshold values of STE parameters associated with IVIG resistance.P = 0.000) and lower global circumferential strain (GCS) . Both GLS and GCS in IVIG‐resistant patients were significantly lower than in IVIG‐responsive patients . ROC analysis demonstrated that the absolute values of GLS < 16.8% and GCS < 15.9% were optimal predictors of IVIG unresponsiveness .Compared with normal children, IVIG‐resistant patients had lower global longitudinal strain (GLS) (15.82 ± 3.32 vs 20.01 ± 2.98, IVIG‐resistant patients presented with more severe LV systolic dysfunction compared with IVIG‐responsive patients, which may be the result of myocarditis rather than CALs. STE may be a helpful diagnostic tool that provides supportive criteria to detect KD patients at a higher risk of IVIG resistance. KD is currently the leading cause of acquired heart disease in children in developed countries.Currently, the most commonly utilized scoring systems for identifying IVIG‐resistant patients are mainly from Japan,22.1In the acute phase (prior to IVIG), we recruited 50 consecutive IVIG‐resistant KD patients and 50 consecutive IVIG‐responsive KD patients who were age‐ and gender‐matched in the First Affiliated Hospital, Xi'an Jiaotong University from January 2016 through April 2018. We also enrolled 50 age‐ and gender‐matched normal children who were referred to our hospital to undergo an echocardiography examination for a murmur without evidence of cardiac abnormalities. All KD patients met the diagnostic criteria of American Heart Association.2.2All the KD patients in the acute phase and normal children underwent transthoracic echocardiography using a commercially available ultrasonic machine  equipped with a S8‐3 transducer. Both the KD patients and normal children were placed in a calm environment for the echocardiographic examination, and subjects who were uncooperative during the echocardiographic examination were sedated with oral hydrate (25‐50 mg/kg). The LV ejection fraction (LVEF) was acquired by biplane Simpson's method of disks, and LV shortening fraction was obtained according to the guideline.2.3The digital images of three consecutive cardiac cycles were obtained and stored for subsequent STE analysis with a commercially available software package . At least 95% of myocardial wall thickness was set to cover. When the trace of the myocardial wall was unsatisfactory, a manual adjustment was used to obtain an adequate speckle tracking image. The width region of interest was set at 5 mm, and the frame rate was set between 60 and 80 frames per second. The zero‐baseline reference point was set at the onset of the QRS complex of the electrocardiogram and was referred to as the LV end‐diastole.2.4To determine intra‐observer variability, STE parameters from 20 randomly assigned patients, including GLS, GLSR, GCS, and GCSR, were reanalyzed by the same observer 2 months after the initial analysis. For inter‐observer variability, the same patients and the same cardiac cycles were analyzed by a second observer.2.5χ2 analysis was used to assess the categorical data differences. The cutoff values of STE measurements associated with IVIG unresponsiveness were determined by receiver operating characteristic curve (ROC) curve analysis. Test reliability was assessed by calculating intra‐observer variability, inter‐observer variability, and intraclass correlation coefficients (ICCs) with a 95% confidence interval.P‐value <.05.Statistical analyses were performed using SPSS 22.0 . Mean and SD were reported for the quantitative echocardiographic data and laboratory data. The categorical data were expressed as the count and percent. The continuous variables were first assessed for normality. For between group differences, the independent‐samples t tests were used for normally distributed continuous variables, and Wilcoxon rank‐sum tests were used for non‐normally distributed continuous variables. The differences in the parameters among IVIG‐resistant group, IVIG‐responsive group and normal group were analyzed using a one‐way analysis of variance with the Student‐Newman‐Keuls test to evaluate the differences between the two groups. A 33.1The age ranges of IVIG‐resistant patients, IVIG‐responsive patients and normal children were 2.2 ± 0.9, 2.2 ± 0.7, and 2.1 ± 0.9 years, respectively. There were 25 males, 27 males, and 27 males in responsive group, resistant group, and normal group, respectively. No significant differences were found in terms of age or gender. In the IVIG‐resistant group, 10 patients with a mild or moderate CALs and two patients with aneurysm were found, whereas in the IVIG‐responsive group, only three patients with mild CALs were found.3.2P = .032) and higher LVMI  compared with normal group. Compared with the IVIG‐responsive group, the IVIG‐resistant group had higher occurrence of CALs , higher LVM  and higher LVMI . In STE analysis, IVIG‐resistant group had lower GLS  and lower GCS  compared with normal group. In addition, the IVIG‐resistant patients exhibited significantly decreased GLS  and GCS   , erythrocyte sedimentation rate (ESR), and C‐reactive protein (CRP) in IVIG‐resistant group were all higher than in IVIG‐responsive group  Table .3.4P = .021) and GCS < 15.9%  were reasonable predictors of IVIG resistance  for GLS, 0.988 (0.969‐0.995) for GLSR, 0.786 (0.547‐0.890) for GCS and 0.995 (0.993‐0.999) for GCSR, respectively. ICCs intra‐observer concordance was 0.973 (0.896‐0.986) for GLS, 0.871 (0.664‐0.956) for GLSR, 0.960 (0.901‐0.985) for GCS, and 0.896 (0.752‐0.965) for GCSR, respectively. Both the inter‐observer and intra‐observer concordances were good for GLS, GLSR, GCS and GCSR values.4Our study showed that IVIG‐resistant patients had both high occurrence of CALs and more serious LV systolic dysfunction than IVIG‐responsive patients. Additionally, STE may assist in differentiating patients with KD at a higher risk of IVIG resistance.Histologic evidence of diffuse myocarditis in the acute phase of KD is present in almost all patientsr = 0.25, P = .0479), suggesting that LVMI is a potential predictor of LV diastolic function and should be added to cardiac function evaluation of KD patients in the acute and subacute phase. In our study, we found that the LVM and LVMI in IVIG‐resistant patients in the acute phase were higher than those in IVIG‐responsive patients and normal children, which indicated that a greater decrease in GLS and GCS in resistant patients compared to responders may be the result of an LV geometry change due to the increased LVM. Therefore, LVM and LVMI should also be regarded as potential predictors of LV systolic function in KD patients. In our study, the inflammatory parameters, including ALB, PLT, ESR, and CRP, in unresponsive patients were all higher than in responders, which also suggested that the myocarditis in IVIG‐resistant patients may be more serious than in IVIG‐responsive patients.Second, myocardial swelling resulting from myocarditis and increased small vessel permeability as a result of coronary arteritis are the main pathophysiology factors of KD patients in the acute phaseThird, in our study, we found that the impaired systolic function in IVIG‐resistant patients may be due to myocardial inflammation rather than the result of coronary arteritis. IVIG‐resistant group in our research had a higher occurrence of CALs than IVIG‐responsive group, which was similar to a previous studyFinally, another finding in our study was that STE may be a helpful technique in detecting KD patients with IVIG resistance. IVIG‐resistant patients have a greater probability of CALs occurrence, and the complications of CALs, including coronary artery stenosis, thrombosis, myocardial ischemia, myocardial infarction, and even sudden death, can occur in the acute phase.This study had several limitations. First, we only assessed KD patients in the acute phase; thus, further studies with a long‐term follow‐up period are recommended. Second, different vendors are not entirely consistent regarding the algorithm of STE, which may restrict comparison among these vendors. Finally, this was only a single center study; therefore multicenter studies are recommended to verify the results.5IVIG‐resistant KD patients had more severe LV systolic dysfunction than patients who were responsive to IVIG. STE is a useful tool for detecting subclinical LV dysfunction in IVIG‐resistant patients. GLS and GCS derived from STE may be helpful diagnostic parameters that provide supportive criteria to detect KD patients at a higher risk of IVIG resistance.The authors declare no potential conflict of interests.


Child sexual offending (CSO) places a serious burden on society and medicine and pedophilia (P) is considered a major risk factor for CSO. The androgen system is closely linked to sexual development and behavior. This study assessed markers of prenatal brain androgenization, genetic parameters of androgen receptor function, epigenetic regulation, and peripheral hormones in a 2 × 2 factorial design comprising the factors Offense (yes/no) and Pedophilia (yes/no) in analyzing blood samples from 194 subjects  matched for age and intelligence. Subjects also received a comprehensive clinical screening. Independent of their sexual preference, child sexual offenders showed signs of elevated prenatal androgen exposure compared with non-offending pedophiles and controls. The methylation status of the androgen receptor gene was also higher in child sexual offenders, indicating lower functionality of the testosterone system, accompanied by lower peripheral testosterone levels. In addition, there was an interaction effect on methylation levels between offense status and androgen receptor functionality. Notably, markers of prenatal androgenization and the methylation status of the androgen receptor gene were correlated with the total number of sexual offenses committed. This study demonstrates alterations of the androgen system on a prenatal, epigenetic, and endocrine level. None of the major findings was specific for pedophilia, but they were for CSO. The findings support theories of testosterone-linked abnormalities in early brain development in delinquent behavior and suggest possible interactions of testosterone receptor gene methylation and plasma testosterone with environmental factors. One major risk factor for CSO is pedophilic preference, which contributes to CSO in about 50% of convicted cases5. Neither the etiology of CSO nor that of pedophilia is well understood. Neurobiological factors have been considered in multifactorial models of both sexual preference and delinquent behavior6.Child sexual offending (CSO) has enormous effects on children’s mental well-being and their development into adulthood10. However, the relationship between androgens and behavior is complex and may depend on different external factors, as well as the age of the organism. As a result, a number of theories have been developed to explain the role of androgens under different circumstances. One of the most important and well examined is organizational–activational theory, which highlights the effects of sex steroids during critical periods of life  leading to sustained dimorphic differentiation of the brain . Activational effects occur when hormones induce acute, sex-specific behaviors in adults by activating or modulating specific brain areas14. In addition, a number of other theories are the subject of discussion, such as the reciprocal model/challenge hypothesis , the basal model (testosterone as a trait marker influencing behavior), and status theory, according to which testosterone facilitates the perception of social status and, e.g., may cause fair bargaining behavior for personal reasons (status seeking)15.Androgens have been ascribed a crucial role in modulating sexual drive and function, as well as violent and aggressive behavior16. Some experts assume very early development during fetal life16, when organizational effects of sex steroids lead to sexual differentiation of the brain. Sexual preference might equally be categorized as sexual orientation, according to some theories19. Regarding the neurodevelopment of sexual orientation, studies have shown that androgens and an X-chromosome-linked inheritance may explain some of the variance20. In their emphasis on prenatal factors, Bao and Swaab go as far as to say, “There is no evidence that one’s postnatal social environment plays a crucial role in gender identity or sexual orientation”16. Clinically, pedophilia occurs almost exclusively in males, which might further support the role of male sex steroids during brain maturation. Although preliminary, small-scale studies on genetic variants in pedophiles (n = 13)21 and paraphilic sexual offenders (n = 97)22 were not able to detect alterations in a specific neurotransmitter system, they were probably underpowered to detect such a link.Only very crude concepts exist of when and how sexual preference might develop24. Another target, cysteine-adenine-guanine (CAG) repeat polymorphisms, has been investigated, with smaller numbers of CAG codon repeats being related to elevated transactivational competence of the androgen receptor protein26. As a consequence, men with shorter CAG repeats may have larger physiological effects of a certain androgen concentration than those of men with longer repeats. Studies have indicated an association of shorter CAG repeats with externalizing behaviors and male violent criminal behavior28.Attempts to better understand the role of androgens in sexual offending have always been challenging. It has become obvious that the relationship between androgens and sexual behavior and aggression is much more complex in humans than in animal models, due to the greater impact of contextual, cognitive, emotional, and social factors. While most of the studies in this area have various methodological flaws, some have indicated a positive correlation between testosterone and/or gonadotropins and antisocial personality traits, novelty-seeking, self-reported hostility, sexual violence, sexual offence recidivism, and impulsive/aggressive behavior in child molesters and/or rapists; for a overview, see refs. All these concepts and initial empirical data show that different biological factors and the time of action need careful consideration when examining the relationship between the androgen system and sexual preference and delinquent behavior. Although we are not able to turn back the clock to explore the etiopathogenesis of sexual preference disorders and sexual offending, this cross-sectional study aimed to provide specific information on prenatal androgenization (by using the marker finger lengths ratio as a proxy), genetic parameters (CAG repeat lengths), gene×environment interactions (methylation status of the androgen receptor gene), and acute measures of the androgen system (plasma concentration of sex steroids and others). This was embedded in the careful clinical selection and characterization of subjects using a 2 × 2 factorial design with the factors pedophilia and offense status, which allowed a distinction to be made between sexual preference and offending behavior.29. NeMUP is a multicenter study in Germany dedicated to disentangling the clinical, neuropsychological, and neurobiological underpinnings of pedophilia and CSO and was funded by the German Federal Ministry of Research and Education (BMBF 01KR1205). For the current analyses, 194 subjects  matched for age and intelligence quotient (IQ) were included. The entire sample consisted of 57 pedophiles with child sexual offenses (P+CSO), 45 pedophiles without child sexual offenses (P−CSO), 20 non-pedophilic subjects with child sexual offenses (C+CSO), and 72 controls (C−CSO).P+CSO, P-CSO and C+CSO were collected via forum posts, email lists as well as from various outpatient departments (German Prevention Project Dunkelfel30) or forensic settings. All participants were comprehensively screened, as described elsewhere29, in order to ensure their appropriate allocation to a specific group according to sexual preference and offense status. The inclusion criterion for pedophilic men was a pedophilic and/or hebephilic preference according to the ICD-10 criteria31 as assessed in a a detailed sexual history together with subjective self-report data from an adapted version of the Kinsey scale for sexual fantasy and behavior32. Allocation to the CSO group (CSO) was based on the history of at least one sexual offence against children under the age of 14 years that involved touching or manipulating  the child’s naked body or genitals with the purpose of sexually stimulating himself, penetrating the child vaginally or anally, or making the child touch or manipulate the offender’s genitals or penetrate him (see for more details ref. 29). Exclusion criteria included intellectual disability, psychotic disorder, current severe major depressive disorder  or anxiety disorder , a clinically predominant substance misuse or dependence, and any psychotropic medication.The sample was part of the NeMUP study, as described elsewhereAll participants provided written informed consent before participating. The five local ethics committees  of the NeMUP research collaboration approved the study.35. The intraclass correlation coefficient between the different raters was 0.716 for the right 2D:4D ratio and 0.664 for left 2D:4D ratio and 0.768 for the total 2D:4D ratio.Flat-bed scans of the left and right hand were obtained using the Canon MP Navigator EX software on a Canon LiDE210 with a resolution of 300 dpi. The distance between the tip of the finger and the ventral proximal crease of the second digit and the fourth digit was measured on black and white DIN A4 printouts by three independent raters and then averagedAll blood samples were drawn immediately before a magnetic resonance imaging (MRI) of the brain as part of the full NeMUP investigation. Time point of blood withdrawal was not significantly different among groups, ensuring comparability and taking into account the circadian rhythms of peripheral sex steroid concentrations. About 19% of blood samples were drawn between 8 and 10 a.m., 17% between 10 and 12 a.m., 7% between 12 a.m. and 2 p.m., 26% between 2 and 4 p.m. and 20% between 4 and 6 p.m. For 11%, the exact time point was missing as no MRI was conducted. Plasma/serum concentrations of total and free testosterone, sex hormone-binding globulin (SHBG), prolactin, and cortisol were measured using enzyme-linked immunosorbent assays  according to the manufacturer’s instructions. Intra-assay and interassay variances were 5.4% and 7.4%, respectively, for total testosterone; 12.3% and 8.8%, respectively, for free testosterone; 9.0 and 8.0, respectively, for SHBG; 4.6% and 5.6%, respectively, for prolactin; 3.5% and 5.0%, respectively, for cortisol. The detection limits of the assays were 0.12 ng/ml for total testosterone, 0.10 pg/ml for free testosterone, 0.77 nmol/l for SHBG, 0.35 ng/ml for prolactin, and 6.79 nmol/l for cortisol.37.Genomic DNA from whole EDTA blood was extracted by means of the QIAamp® DNA Blood Mini Kit , according to the manufacturer’s protocol on a Biomek Nxp . For the analysis of methylation rates, 500 ng genomic DNA from every sample was bisulfite-converted using the EpiTect® Bisulfite Kit . Using this procedure, unmethylated cytosines are converted into uracils via deamination (after polymerase chain reaction (PCR): thymins), whereas methylated cytosines are protected from alteration40. For sequencing in accordance with Sanger, 30 ng of the PCR product and AR-F were utilized in the BigDye Terminator v3.1 Cycle Sequencing Kit . The following Sequencing Standard Protocol was used for sequencing PCR: 96 °C for 1 min, 28 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Afterwards, samples were kept at 12 °C in the instrument until further processing.The following primers were used for amplification of the CAGn androgen receptor polymorphism: AR-F (forward) 5′-CGCCGTCCAAGACCTACC-3′ and AR-R (reverse) 5′-GAACCATCCTCACCCTGCT-3′A fragment of 414 base pairs (from −98 to +316 bp) containing 27 CpG-sites was analyzed, covering part of a big CpG-island reaching from position −204 to +666 as defined by the “Geneious” software. Oligonucleotide primers used for the amplification PCR had the following sequences: 738_Arz_Bis_Fw: AGGGAAAAGGAGGTGGGAA (forward primer) and 784_Arz_Bis_Rev: AAATAAAAAAAAAAAAAACAAAAACAAC (reverse primer). The corresponding touchdown-PCR protocol was composed as follows: the initial taq activation step (15 min at 95 °C) was followed by 15 cycles of denaturation (30 s at 95 °C), primer annealing , and elongation (72 °C for 40 s). Afterwards, 25 cycles followed with a denaturation temperature of 95 °C for 30 s, annealing at 54 °C for 40 s, and elongation at 72 °C for 40 s. In the end, samples were incubated at 72 °C for 4 min and kept at 12 °C until further processing or storage in the refrigerator. For sequencing PCR, again the BigDye Terminator v3.1 Cycle Sequencing Kit  was used with the forward primer and roughly 30 ng of the amplification PCR product. For sequencing PCR, the protocol “SeqATRev” was used, which entailed 96 °C for 1 min, followed by 25 cycles of 96 °C for 5 s, 60 °C for 90 s, and 50 °C for 90 s. Again, samples were kept at 12 °C in the instrument afterwards.All sequencing was done on a Genetic Analyzer 3500 xL (Applied Biosystems).41. Exclusively CpG sites with valid results in >70% of all samples were used for the analysis.Sequences were analyzed using the ESME Software (ESME). ESME determines the DNA methylation levels of sequence trace files and additionally performs quality control, normalization of signals, and correction for incomplete bisulfite conversion. Alignment of generated bisulfite and the respective reference sequences enables comparison of C to T values (in the forward sequence) and G to A values (in the reverse sequence) at CpG sitesp < .05 were considered statistically significant. After testing for normal distribution, demographic, clinical, and endocrine data was analyzed using a 2 × 2 analysis of variance (ANOVA) (preference×CSO) with age and IQ as covariates. CAG repeat length data were pooled for analysis into three subgroups, resulting in subjects with short (7–18), medium (19–23), or long (24–32) CAG repeat length42. For methylation analysis, CpG positions with a variance of <0.001 over the whole sample were excluded from further analysis. Through this procedure, two CpG positions were discarded (−054 and +068). We then performed a linear mixed models analysis using a restricted estimated maximum likelihood approach with two hierarchical levels subjects and the repeated measure within-subject factor CpG site over the remaining 25 CpG positions. DNA methylation was computed as the dependent variable and CpG site, CAG repeats, pedophilia, and offense status as fixed factors. Interactions between CpG site and other factors, as well as between CAG repeats and other factors in the model, were also assessed. The relation between methylation and hormones was analyzed by correlating mean methylation over the whole analyzed sequence and concentrations of hormones.Statistical analysis was performed using IBM SPSS Statistics 22 . Results with a F = 5.537, p = .02, η2 = 0.03), and elevated levels of own perceived traumatization and neglect during childhood as assessed by the Childhood Trauma Questionnaire  = 29.88, p < .001, η2 = 0.14; interaction effect between offender and pedophilia  = 7.33, p = .007, η2 = 0.038; Table The four groups did not differ in age (range 36.8–40.5 years) or intelligence (estimated IQ between 93.8 and 103.2). Clinical examination revealed significantly higher levels of impulsivity as assessed by the Barret Impulsivity Scale  in child sexual offenders  = 4.84, p = .029, η2 = 0.027) and borderline effects for free testosterone  = 2.96, p = .087, η2 = 0.017; Table F = 8.04, p = .005, η2 = 0.045). Finally, there was a marginally lower testosterone/cortisol ratio in child sexual offenders compared with non-offending subjects  = 3.39, p = .067, η2 = 0.019; Table In order to examine the different hypotheses on the role of sex and corticosteroid hormones in regulating behavior as outlined above, different endocrine parameters were analyzed. These analyses revealed lower testosterone levels in child sexual offenders for total testosterone  = 6.41, p = .012, η2 = 0.037), as well as a trend toward smaller 2D:4D ratios of the right hand  = 3.7, p = .056, η2 = 0.022). For left hand 2D:4D ratios and mean 2D:4D ratios (mean of both hands), interaction effects were found  = 5.53, p = .02, η2 = 0.032; mean: F = 4.35, p = .039, η2 = 0.026)  of 2D:4D digit ratio in child sexual offenders (6) Table .Table 3RF = 6.02, p = .014), CpG position  = 4.28, p < .001), and CAG repeat length  = 6.94, p = .001) on methylation levels  = 3.48, p = .031; Fig. F = 4.08, p = .017; Fig. As in previous studies, we use a linear mixed model analysis to explore the relationship between epigenetic and genetic markers with offense status and sexual preference. These analyses revealed main effects of offense status .Bonferroni corrected post hoc r = −.204, p = .03), while neither the whole sample  nor the offending group  showed this correlation.In addition, a negative correlation between CAG repeat length and methylation levels was found only in the non-offending group , left hand , and mean of left and right hand . Moreover, the total number of sexual offenses was positively correlated with mean methylation of the androgen receptor gene .To evaluate the behavioral aspects associated with methylation of the androgen receptor, correlations between methylation and clinical characteristics, hormone levels, and prenatal androgenization were accessed. Correlational analyses revealed a significant inverse association between the total number of sexual offenses and 2D:4D ratios of the right hand  than for left hand ratios53. Intriguingly, these proxy markers were correlated with total number of child sexual offenses, which may further support the role of prenatal androgenization in the development of delinquent behaviors.With regard to previous empirical data and models, the current data set fits the conception of a prenatally more masculinized brain (assessed here by a proxy marker) in CSO, prone to externalizing behaviors and disordersIn terms of genetic factors, there were no main effects of androgen receptor polymorphism (CAG repeat lengths) on either offense status or sexual preference at a group-level analysis (ANOVA). However, mixed models analyses indicated significant effects of CAG repeat lengths for the extent of androgen receptor gene methylation. On the one hand, shorter CAG repeats were associated with a higher percentage of methylation Fig. . On the Finally, correlational analyses revealed a negative association between markers of prenatal androgenization and number of child sexual offenses, as well as a positive association between methylation level and total number of child sexual offenses. Thus one might speculate that an elevated prenatal androgenization of the brain compromises the regulatory abilities of the androgen system to adapt to environmental factors in later life.Although some of these results warrant replication because of the small sample size of the subgroups, it might be speculated that higher levels of methylation are an attempt by the organism to downregulate a highly functional androgen (receptor) system and/or downregulate androgen-driven impulsivity and aggressive behaviors toward children in those subjects who have committed CSO in the past but are currently willing to control their behaviors. As mentioned below, our samples consisted predominantly of men recruited from outpatient departments and forensic settings, most of whom were motivated not to reoffend. In line with our interpretation, peripheral concentrations of plasma testosterone and testosterone/cortisol ratios were lower in offending than in non-offending subjects, which may reflect a restrictive and well-controlled coping strategy.55.Comparative studies exploring the interaction between organizational and activational effects of the androgen system are scarce or even non-existent. Previous studies of normal men have failed to find an influence of the androgen receptor gene on right hand 2D:4D ratio, with men with more sensitive androgen receptors even tending to score lower on subscales of aggression questionnaires57. However, most of them failed to distinguish between sexual preference and offense status, making it difficult to draw conclusions regarding the origins of pedophilia and/or offending behavior. Only recently, the NeMUP consortium has taken into account the differential effects of these two factors. This has produced a series of results indicating more normal than abnormal findings regarding the neuropsychological and neurobiological factors underlying pedophilia. Most importantly, the majority of findings were attributable to CSO and included worsened response-inhibition abilities58 and lower right temporal pole volumes in offending pedophiles59, as well as superior inhibitory control (Go/No-Go paradigm) accompanied by inhibition-related activation in the left posterior cingulate and left superior frontal cortex in non-offending pedophiles60. These findings, together with our current findings on the androgen system, can be implemented in a working model of neurobiological factors contributing to CSO  was sufficiently large to draw conclusions.29 was carried out which revealed higher levels of impulsivity and experienced neglect and traumatization in child sexual offenders compared to non-child offenders. However, apart from the number of offenses, which actually showed a very intriguing association with prenatal and epigenetic parameters, other measures showed no significant associations. Additional parameters on, e.g., direct and indirect aggression, as well as other static and dynamic risk factors, might have led to additional results that were not apparent in this data set.Careful clinical and psychometric examination of these subjects49). Additionally, data on metabolites of testosterone in the brain, such as estradiol and dihydrotestosterone, and their respective enzymes aromatase and 5α-reductase, were not assessed and could be of value in future analyses.The assessments carried out here provide only a snapshot of the role of the androgen system and thus may not entirely reflect what has happened in the past or is currently happening in these subjects. Proxy markers such as 2D:4D finger length ratios may be helpful, but they can be affected by many factors and diseases and therefore may be rather non-specific or of questionable reliability .Many participants were drawn from outpatient departments or forensic settings, which might have led to a bias in our sample toward people who seek help and show cooperation, thereby directly affecting epigenetic and endocrine processes.Taken together, these initial results may point toward the importance of the androgen system for a better understanding of the different pathways toward sexual violence. Prenatal and epigenetic measures combined with a careful clinical characterization appear promosing tools for future studies, which may also focus on different forms of sexual violence, e.g., against children vs. against adults.


Culicoides species from the Obsoletus group are important vectors of bluetongue and Schmallenberg virus. This group consists of several species that cannot easily be identified using morphological characteristics. Therefore, limited information is available about their distribution and habitat preferences. In this study, we aimed to elucidate the species composition of the Obsoletus group in three habitat types at climatically different latitudes across Europe. Traps were placed in three habitat types in three countries at different latitudes. After DNA extraction, biting midges were identified using PCR and gel electrophoresis. Extraction of DNA using Chelex proved to be a cost and time efficient method for species identification. A latitudinal effect on the relative abundance of species from the Obsoletus group was found. Species composition was unique for most country‐habitat combinations. The majority of biting midges were either C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats. Their wide distribution and their high abundance at livestock farms make these species likely candidates for rapid farm‐to‐farm transmission of pathogens throughout Europe. Our results emphasize the need to differentiate Obsoletus group species to better understand their ecology and contribution to pathogen transmission. The relative abundance of species from the Obsoletus group was more defined by latitude than by habitat.C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats.The majority of biting midges from the Obsoletus group were either Extraction of DNA from biting midges using Chelex proved to be a cost and time efficient method for species identification. Culicoides imicola  was responsible for virus transmission. While C. imicola was known to transmit bluetongue virus in African and south‐European countries , C. dewulfi , C. montanus , C. obsoletus sensu stricto (s.s.) , and C. scoticus   have been found breeding in a much wider range of substrates  and C. dewulfi prefer the head, back, and flanks  in three countries  representing three different latitudes in Europe. For this purpose, we employed a controlled study design with the same sampling effort across all locations. We expected species from the Obsoletus group to have specific ecological preferences for larval habitats, hosts, as well as climate. Therefore, we hypothesized that each habitat and country would represent a unique composition of Obsoletus group species.Collection and identification of biting midges was earlier described by Möhlmann et al. . In shorCulicoides biting midges were identified to species level with the use of the Interactive Identification Key for Culicoides (IIKC)  belonged to the Obsoletus group  was used based on an earlier identification protocol of Obsoletus group species , C. obsoletus s.s. obs‐COI‐fwd (5'‐CAGGAGCTTCTGTAGATTTGGCT‐3′), C. scoticus sco‐COI‐fwd (5′‐CCACAATTATTAATATGCGATCTACC‐3′), and C. chiopterus chio‐COI‐fwd (5'‐CCTTTATTTGTTTGGTCTGTTCTTC‐3′) were used. The mastermix for one sample consisted of 5 μL of 5X colourless reaction buffer, 6 μL of MgCl2 (3 mM), 5 μL dNTPs (1 mM), 2 μL of the forward primer (10 μM), 0.5 μL of every reverse primer (10 μM), 0.125 μL GoTaq polymerase (5 U/μL) , 3.375 μL MilliQ, and 3 μL target DNA obtained from DNA extraction. The total volume of 25 μL was used for amplification with PCR settings on 15 min at 94°C, followed by 42 cycles of 30 s at 94°C, 45 s at 63°C, 45 s at 72°C, and a final step of 5 min at 72°C. Final temperature was kept at 4°C until samples were stored in the freezer at −20°C before further use.For differentiation among species within the Obsoletus group the protocol as described by Lehmann et al.  was usedC. dewulfi was 468 bp, C. obsoletus s.s. 318 bp, C. scoticus 237 bp, and C. chiopterus 190 bp  were mixed with Orange G loading dye (5 μL) and loaded on a 1.5% agarose gel for electrophoresis for 45 min at 80 V. A 100 bp ladder was used as reference, as well as a negative control and positive controls for each of the four species. After electrophoresis, the gel was exposed to UV light in a Bio‐Rad Gel Doc and imported into computer program Quintify One to visualise the bands. Species were identified according to differences in PCR product length whereby https://blast.ncbi.nlm.nih.gov). All COI samples were confirmed with at least 99% identity. The DNA extracts of these samples were diluted 1:1 and used as a positive control for the rest of the PCR identifications.The first PCR products from the expected four species of the Obsoletus group were excised from the agarose gel. They were subsequently recovered by the QIAquick Gel Extraction Kit (Qiagen), and sent to Eurofins Genomics  for sequencing. The received COI sequences were assembled with Geneious and tested with the use of Nucleotide BLAST against the NCBI GenBank database  on the ratios of C. chiopterus, 43 (6.8%) as C. dewulfi, 327 (52.1%) as C. obsoletus s.s., and 189 (30.1%) as C. scoticus. The remaining 27 (4.3%) biting midges did not yield a PCR product (S1 Dataset).Of the 628 selected biting midges from the Obsoletus group, 42 (6.7%) were identified as 2 = 122.69, df = 6, P < 0.001; Fig. 2 = 36.03, df = 6, P < 0.001; Fig. P < 0.001). In Sweden, we found three species of the Obsoletus group , compared to four in The Netherlands, and only the two species of the Obsoletus complex (C. obsoletus s.s. and C. scoticus) in Italy. Wetlands had relatively more Obsoletus complex biting midges, and less C. chiopterus and C. dewulfi, when compared to both farms (P < 0.01) and peri‐urban habitats (P < 0.001). There was no significant difference in ratios between farms and peri‐urban habitats (P = 0.18).The ratios of species were significantly different among the three countries . In Italy, only farms were different from the wetland habitat (P < 0.05), due to relatively high proportions of C. scoticus at farms and high proportions of C. obsoletus s.s. in wetlands  and Italy (P < 0.05). In The Netherlands the four Obsoletus group species had a more even distribution, differentiating this country from the other countries in all habitats (P < 0.01). In Italy, relatively high proportions of C. obsoletus s.s. were recorded in all habitats, except when compared with Swedish farms which had relatively more C. obsoletus s.s. biting midges than Italian farms (P < 0.05).Ratios of the species were significantly different among countries within each of the three habitats  occur in the three selected European countries in relatively high proportions. The other two species from the Obsoletus group were not found in each of the studied countries. Culicoides dewulfi was found in collections from both Sweden and The Netherlands, while C. chiopterus was only found in collections from The Netherlands. Neither of these two species were recorded in the collections from Italy. These results are in line with previous findings in Sweden, The Netherlands, and Italy that demonstrated that the two species of the Obsoletus complex are the dominant species in the Obsoletus group across Europe  is limited. In one case, for example, only nine Obsoletus group specimens were collected during a 1‐year sampling effort (peri‐urban habitat in Sweden). In addition, the study was carried out in only one region per country. These points emphasize that our collections were local, and cannot easily be extrapolated to country level.Molecular identification of many samples can be labour intensive and relatively expensive. In this study we show that alternative methods can be used. We first used the DNeasy Tissue Kit for DNA extraction, as described by Lehmann et al. . We thenC. montanus, as specific primers for this species were not included in the PCR. However, thus far C. montanus has only been identified in relatively low numbers in southern Italy  did not yield a PCR product. These could represent failed DNA extractions, or they could include uncharacterized new species  species are being identified within the Obsoletus group  was found at all latitudes, C. chiopterus was only identified in samples collected in The Netherlands, whereas C. dewulfi was not found among samples originating from Italy. Habitat types also influenced the ratios of species within the Obsoletus group, but effects were not consistent at different latitudes. Our suggestion to use a more efficient method for identification of Obsoletus group species may encourage others to also perform species identification, so that this becomes routine practice for Culicoides studies. The majority of the biting midges identified was part of the Obsoletus complex (82.2%), and both species were found at all latitudes and in all habitats. Their known susceptibility to viruses in combination with their wide distribution and high densities at livestock farms make C. scoticus and especially C. obsoletus s.s. likely candidates for rapid spread of midge‐borne viruses throughout Europe.A strong country effect, indicative of latitudinal effects, on the relative abundance of species from the Obsoletus group was found. While the Obsoletus complex (The authors declare that there is no conflict of interest.Table S1. Dataset. Primary data of selected and analysed Obsoletus group biting midges.Click here for additional data file.


Our early study has found valproic acid (VPA)‐induced lipid dysmetabolism in animal model, however, the details of lipid profiling of VPA‐treated epileptic patients remain unknown. Therefore, in this study, the blood samples of VPA‐treated epileptic patients and VPA‐free controls were collected for lipidomic and biochemical assays. As results, clinical data showed the changes of some blood lipid molecules in VPA‐treated epileptic patients. In lipidomic assays, all 3797 annotated positive ions were identified prior to the data validation. In addition, the number of differentially expressed lipids were identified. And the 133 lipid molecules in VPA‐treated cases were significantly up‐regulated when compared to those in controls, while other 250 lipid metabolites were down‐regulated. Further, these lipid metabolites were mainly constituted with glycerolipids, glycerophopholipids, fatty acyls, sterol lipids. In addition, the most significant elevations of metabolite molecules of triglyceride, sphingomyelin, phosphorylcholine, ceramides, phenolic phthiocerol, as well as topped reductions of phosphoethanolamines, diradylglycerols, 1α,25‐dihydroxy‐24‐oxo‐22‐oxavitamin D3, 2‐deoxy‐20‐hydroxy‐5alpha‐ecdysone 3‐acetate, dolichyl‐4 phosphate were identified respectively. Taken together, these clinical findings demonstrate that negative impacts of exposure to VPA on expression of lipid mediators, progressively disrupting the functions of lipid molecules. Interestingly, these differentially expressed metabolites may be potential biomarkers for screening VPA‐induced dyslipidemia. In clinical screening, other chronic diseases and metabolic disorders among these cases could be excluded through biochemical check‐up and clinical follow‐up. In addition, three adults with healthy conditions were set as a control group. Briefly, the blood samples of all subjects were isolated for plasma preparation. And parts of samples were used for lipidomic tests, while others were employed in instrument analysis and biological assays. The human protocols were implemented strictly on the basis of the Ethical Guidelines of the Declaration of Helsinki.2.2Methodologically, the epileptic plasma samples were deproteinized by using commercially available reagents. To begin with, internal standards of VPA  were prepared for different doses to plot a standard curve. The equal volumes of samples and standards were automatically added to a high performance liquid chromatography (HPLC) system  equipped with an SIL‐20AC detector and injector for VPA determination. The VPA separation was achieved by using an ASTON RG C18 column , followed by maintaining constant temperature 45°C in the column. In addition, the working mobile phase (acetonitrile:isopropanol:purified water = 23:8:69) was set as 1.1 mL/min flow rate. The VPA peak area was determined in comparisons of retention time (RT) of tested samples and internal standards. As results, the quantitative contents of plasma VPA were identified through a standard curve.2.3g, the supernatants were transferred to another 96‐well plate. All samples were obtained by the liquid chromatograph‐mass spectrometer (LC‐MS) system following machine orders. The chromatographic separation was conducted by using an ultra‐performance liquid chromatography system . A chromatographic column  was applied to the reversed phase separation. The flow rate was maintained at 0.4 mL/min with mobile phase of solvent A and solvent B. A high‐resolution tandem mass spectrometer TripleTOF5600plus  was employed to identify the metabolites form column. The data of mass spectrometry were collected following the IDA mode, and the TOF molecular weight ranged from 60 to 1200 Da. In data collection, the mass accuracy was calibrated every twenty samples. In addition, in an attempt to assess the LC‐MS stability, a quality control (QC) sample was identifiable after every ten samples.Firstly, the plasma metabolites were extracted with 50% methanol buffer. After being centrifugated at 4,000 2.4P‐value was produced for multiple tests by using a false discovery rate (Benjamini‐Hochberg). Partial least squares‐discrimination analysis was assessed to differentiate the possible variables between groups. Further, a cut‐off value was applied in identification of all core features.Each positive ion was screened through pooling RT and mass charge ratio. Intensities of detectable peaks were recorded, and all information of a three dimensional matrix with peak indices and ion intensity was produced. The web‐accessible databases of Kyoto Encyclopedia of Genes and Genomes, Human Metabolome Database were employed to annotate the metabolites through correlating the accurate molecular mass data. If a mass difference of database value was <10 ppm, the identifiable metabolites would be annotated and the molecular formula would be further validated through the isotopic distribution determination. Additionally, an in‐house fragment spectrum library of metabolites was used to validate the identifiable metabolites. Those detectable features with <50% of QC samples or 80% of biological samples were excluded, other remaining peaks were evaluated with the k‐nearest neighbor algorithm to further enhance the quality of data. The principal components analysis (PCA) was implemented for outlier inspection and batch impacts assessment by using the pre‐processed dataset. Additionally, the relative standard deviations of the metabolic features were measured from total QC samples, and those data >30% were excluded. Wilcoxon test was performed to analyze differences in metabolite contents between 2 phenotypes. The 2.5t test. Groups were considered to be significantly different if a P < 0.05. Result was expressed as mean ± SD.Statistical assessment was processed through statistical product and service solutions 19 . Fold‐change ratio of differentially expressed metabolites between two groups were analyzed by Student's 33.1In medical imaging inspection, these epileptic cases showed certain brain impairments, mild leukoaraiosis, brain atrophy. And other key internal organs  with normal morphologies were observed in ultrasonic inspection Figure . Results3.2In preliminary lipidomic statistics, a total of 3797 annotated positive ions were identified. And the number of candidate metabolite Figure A, lipidm3.3To screen statistically and biologically significant lipid molecules from a large number of detectable metabolites, further analyses aimed to reveal the changes of metabolic processes and mechanisms following VPA treatment in epileptics. In ratio evaluation, relative low levels of experimental errors in fold‐change of lipids percentage in all groups were testified Figure A. In PCA4Valproic acid, a medical antiepileptic, is found with induction of lipid‐associated dysmetabolism over long‐time treatment.Metabolomics refers to a scientific method for disclosing all biological fingerprints associated with metabolites, molecule intermediates, and products of metabolism.P < 0.05. Thus, top 133 up‐regulated and 250 down‐regulated lipid metabolites were identified. In addition, the detailed information of all these differential expressed lipids were listed. And the top 5 elevated metabolites of triglyceride, sphingomyelin, phosphorylcholine, ceramides, phenolic phthiocerol, as well as top 5 reduced metabolites of phosphoethanolamines, diradylglycerols, 1α,25‐dihydroxy‐24‐oxo‐22‐oxavitamin D3, 2‐deoxy‐20‐hydroxy‐5alpha‐ecdysone 3‐acetate, dolichyl‐4 phosphate were identified respectively. Taken together, we reasoned that top differential expressed lipids may contribute to development of alternative diagnosis for VPA‐induced metabolic disorders.In further quantitative determination of these lipid metabolites, the higher quality features of 7700 lipids were identified, followed by quantitative QC. All testing methods showed the better credibility and repeatability of these identifiable data. In addition, in order to screen and characterize the differential expressed lipids of VPA‐treated epileptics, all testing lipid metabolites were further re‐assayed through designed criteria with fold‐change ratio higher than two and 5In brief, the clinical findings elucidate that VPA‐treated epileptic patients may induced brain impairment‐related dyslipidemia. Interestingly, lipidomic approach can contribute to identification of differential expressed lipid metabolites, and these molecules may be the potential markers for screening VPA‐induced dyslipidemia.The authors declare that there are no conflicts of interest. Click here for additional data file.


The current staging systems do not consider the tumor location of thymomas, and its clinical relevance is poorly understood. This study aimed to evaluate the impact of tumor location on the clinicopathological features and prognosis of thymomas.We performed a retrospective review of patients at our institution who underwent an extended thymectomy for a thymoma from 1976 to 2015. The tumor location was classified as either the superior or inferior mediastinum based on the maximum tumor diameter. The clinicopathological characteristics of the thymoma were also evaluated. Kaplan‐Meier estimates and Cox proportional hazards models were used to analyze the survival outcomes and risk factors for recurrence.n = 167), the superior mediastinum group (n = 27) had a higher frequency of myasthenia gravis (MG), advanced Masaoka‐Koga staging, disease progression and recurrence (P < 0.05). The Kaplan‐Meier analysis demonstrated thymomas in the superior mediastinum had worse survival outcomes that included overall survival, progression‐free survival and disease‐free survival (P < 0.05). The multivariate analysis showed tumor location was an independent prognostic factor for all the survival outcomes (P < 0.05). Furthermore, the tumor location (P = 0.004) and Masaoka‐Koga stage (P < 0.001) were the only two independent risk factors for recurrence in the multivariate analysis.A total of 194 patients with thymoma were eligible for this study. Compared with the inferior mediastinum group (The clinicopathological features of thymomas on MG, Masaoka‐Koga staging, disease progression, and recurrence were different between locations of superior and inferior mediastinum locations. Thymomas in the superior mediastinum tended to be associated with worse survival and increased recurrence. This retrospective review included all the patients at our institute with a thymoma who underwent an extended thymectomy from 1 January 1976 to 31 December 2015. The clinical information was retrieved from electronic (2000–2015) and archived paper charts (1976–1999). The inclusion criteria for patients in this study were as follows: (i) a pathologically confirmed thymoma; (ii) complete clinical and pathological data accessible for restaging based on the Masaoka‐Koga stageThe boundaries of the superior mediastinum were defined by previous publications as the level between the sternal angle to the inferior margin of the fourth thoracic vertebrae.The postoperative follow‐up interval was every 3–6 months, and chest X‐ray or computed tomography (CT) were performed annually. Any other necessary examinations were determined by the patient's clinical needs. The final follow‐up visit was in October 2018 to evaluate survival and recurrence outcomes.t‐test, chi‐square test, linear‐by‐linear association, likelihood ratio detection or Fisher's exact test. The OS and PFS were evaluated using the Kaplan‐Meier method. The differences between survival curves were analyzed by a log‐rank test. Furthermore, patients who only underwent an R0 surgery were selected to evaluate DFS with the same methods described above. To assess the significance of tumor location as an independent prognostic risk factor for OS, PFS and recurrence, univariate and multivariate analysis were performed using the Cox's proportional hazards model. The hazard ratio (HR) was estimated with a 95% confidence interval (CI) for both the univariate and multivariate analysis. Since only a subset of the patients were comorbid for MG and underwent a lymph node dissection, we did not include the MGFA classification or lymph node metastasis as covariates in the univariate or multivariate analysis. The following variables were finally included as the starting set of covariates for the univariate analysis: tumor location (Superior/Inferior), age (as continuous), tumor diameter (as continuous), sex , MG (Yes/No), anti‐AchR (positive/ negative), WHO histologic classification (A/AB/B1/B2/B3), Masaoka‐Koga stage (I/II/III/IV), MPMT (Yes/No), surgical radically (R0 resection/non‐R0 resection), lymph node dissection (Yes/No), preoperative induction therapy (Yes/No), and postoperative adjuvant therapy (Yes/No). Only variables with a P < 0.1 in the univariate analysis were included as covariates for the multivariate analysis. Two‐sided tests were applied and a P < 0.05 was considered to be a statistically significant difference. The statistical analysis was performed using SPSS 24.0 .Demographic and clinicopathologic variables were collected and included in the statistical analysis. The descriptive statistics are reported as the mean ± standard deviation. The categorical variables are reported as frequencies and proportions. The differences between the groups of superior and inferior mediastinum were assessed using a Student's P = 0.811), respectively. Patients with thymomas in the superior mediastinum group (n = 27) accounted for 13.9% of the total cases while those with thymomas in the inferior mediastinum group (n = 167) accounted for 86.1%. The clinicopathological characteristics of patients with superior and inferior mediastinum thymomas are shown in Table P = 0.811, 0.448, 0.881, 0.492, 0.134, 0.069, 0.382, 0.721, 0.881, 0.268, 0.114, and 0.998, respectively). However, MG, Masaoka‐Koga stage, disease progression and recurrence were significantly different between the two groups . More patients with thymomas in the superior mediastinum had MG (55.6% vs. 29.3%), Masaoka‐Koga stage III/IV disease (40.7% vs.18.6%), and disease progression (44.4% vs. 8.4%) than those with thymomas in the inferior mediastinum. Only patients who underwent an R0 resection were included to evaluate recurrence. Patients with thymomas in the superior mediastinum tended to have a higher frequency of recurrence (37.5% versus 7.2%) than those with thymomas in the inferior mediastinum (P < 0.001).A total of 194 patients with thymomas were eligible to be included in this study, including 98 males and 96 females with a mean age of 53.2 ± 13.5 years versus 53.9 ± 14.7 years . Patients with thymomas in the superior mediastinum experienced a significantly worse OS and PFS than those with thymomas in the inferior mediastinum. The Kaplan‐Meier curves are shown in Figures The median follow‐up period was 115.0 months (range: 0–399 months). The OS and PFS were 188.5 ± 21.4 months and 133.7 ± 23.2 months in the superior mediastinum group versus 349.3 ± 11.7 months and 316.2 ± 15.4 months in the inferior mediastinum group, respectively. The differences in OS and PFS between the superior and inferior mediastinum groups were significant (P < 0.001). Thymomas in the superior mediastinum were associated with significantly worse DFS. The Kaplan‐Meier curves are shown in Figure DFS was also assessed in this study, but only with patients who underwent an R0 resection. The DFS for patients with superior and inferior mediastinum were 149.3 ± 24.5 days and 330.4 ± 13.5 days, respectively. There was a significant difference in DFS between the two groups . In addition, the tumor diameter, WHO classification and surgical radicality were shown to be predictors of PFS in the univariate analysis (P < 0.05). In the multivariate analysis, only tumor location and age were found to be independent prognostic factors of OS  and PFS . Thymomas in the superior mediastinum were associated with a worse OS  and PFS . Additionally, preoperative induction therapy was shown to be an independent prognostic factor for OS (P = 0.030), but not for PFS (P = 0.466). The Masaoka‐Koga stage was an independent prognostic factor for PFS (P = 0.004) but not for OS (P = 0.272). In contrast, sex, MG, anti‐AchR, MPMT, lymph node dissection, and postoperative adjuvant therapy were not prognostic factors in either the univariate or multivariate analysis (P > 0.05). The results are presented in Tables In the univariate analysis, both OS and PFS were associated with tumor location, age, Masaoka‐Koga stage and preoperative induction therapy (P < 0.05), while tumor location  and Masaoka‐Koga stage  were the only independent risk factors identified in the multivariate analysis. Recurrence was more likely for thymomas in the superior mediastinum and advanced Masaoka‐Koga stages than for thymomas in the inferior mediastinum (Table Only the patients with an R0 resection were included in the present analysis of recurrence. For the 176 patients, tumor location, tumor diameter, WHO classification, Masaoka‐Koga stage and preoperative induction therapy were identified as significant variables by the univariate analysis (It is particularly important to determine the specific characteristics of thymomas. However, no previous studies have discussed the importance of tumor location in determining clinicopathological features or its relationship to prognosis. While many authors have attempted to identify prognostic factors and risk factors for the recurrence of thymomas, none have identified tumor location as a risk factor.et al. 33.0% of patients with thymomas had MG, which was similar to the incidence observed in the ITMIG database.In the study by Padda Masaoka‐Koga stage III/IV accounts for approximately 30% of all patients with thymomas.Progression and recurrence after the initial thymoma is resected is observed in 6.9%–18.0% of patients and can occur even decades after the primary resection.When compared to tumors located in the inferior mediastinum, the thymomas of the superior mediastinum constituted a small proportion of the thymomas in this case series. However, the patients of the superior mediastinum group had an overall worse prognosis according to our survival analysis, and thus, their thymomas may have been more aggressive in nature. The OS, PFS and DFS in the inferior mediastinum group were largely consistent with previous reports, but those in the superior mediastinum group were worse than what has been previously reported.It is difficult to determine a clear set of prognostic factors for thymomas due to the excellent patient survival. A review of prognostic risk factors for thymomas summarized 29 studies that used multivariate analysis to determine risk factors for survival and showed that the Masaoka‐Koga stage was the most important factor associated with survival outcomes, followed by the WHO classification, surgical radicality and tumor size.The ITMIG established that the tumor recurrence rates are the best measure of treatment efficacy in thymomas from at any site.The results from a prospective multicenter database identified associations between higher Masaoka stage and increasing tumor size with increased recurrence rates. Sex, WHO classification, and adjuvant therapy were not risk factors for recurrence.Collectively, these results demonstrate that tumor location should be included in the future staging systems for thymomas. Furthermore, there might be differences in the optimal treatment protocols for superior and inferior mediastinum thymomas, such as induction treatments, adjuvant therapies and follow‐up intervals. Future multicenter and prospective studies are necessary to confirm this new proposal.There were several limitations of this study. First, this was a retrospective study with data that was collected over a period of approximately 40 years, which may have led to different diagnoses from the pathologists and surgeons. Second, the preoperative imaging data generated prior to 1999 could only be collected from chart records, which may be less accurate for distinguishing tumor locations than the information obtained directly from electronic records. Third, we could not include every potential confounding variables in our multivariate model. Additional multicenter prospective studies of the primary tumor location are required to confirm our findings and improve insights into the staging and treatment of thymomas.In conclusion, this is the first report to demonstrate the significant impacts of tumor location on a range of clinicopathological characteristics and prognosis in patients with thymomas. The clinicopathological features of MG, Masaoka‐Koga stage, disease progression and recurrence were different between the superior and inferior mediastinum thymomas. Importantly, we showed that thymomas in the superior mediastinum were associated with worse survival and more recurrence.The authors have no conflicts of interest to disclose.