The aim was to evaluate the efficacy and safety of different combination strategies for prophylaxis of venous thromboembolism (VTE) after gynecologic surgery in patients at different levels of risk. This was a prospective multicenter randomized controlled study, in which 625 women who would undergo pelvic surgery for gynecologic diseases were stratified into three risk groups and then randomized into four groups to receive graduated compression stockings (GCS) alone (group A), GCS + low molecular weight heparin (LMWH) (group B), GCS + intermittent pneumatic compression (IPC) (group C), and GCS + IPC + LMWH (group C), respectively. The overall incidence of DVT was 5.1%. Group A had the highest incidence of DVT (8.8%), followed by group C (5.2%), group B (3.8%), and group D (2.6%). There was a significant difference in the incidence of DVT between groups A and D. The incidence of DVT was significantly lower in LMWH-treated patients (group B + group D) than in non-LMWH-treated patients (group A + group C). In conclusion, combination prophylaxis, especially LMWH-containing strategies, is better than monoprophylaxis in reducing VTE after gynecologic surgery. Risk-stratified prophylactic strategies should be implemented in patients undergoing gynecologic surgery, with LMWH-containing strategies being recommended for high-risk and very-high-risk patients. Epidemiological studies from our team and other groups show that the incidence of DVT following gynecologic surgery ranges from 6.2% to 37.9%4. Although the true incidence of PE in patients after gynecologic surgery is currently unknown, it has been reported that 0.3–4.4% of patients undergoing thromboembolic prophylaxis after gynecologic oncology surgery developed PE6. PE is the leading cause of mortality following gynecologic surgery, with a mortality rate as high as 40%2. Since the majority of patients with fatal PE often die within 30 min following the appearance of symptoms, there is no chance of thrombolytic therapy or surgery. Therefore, the prevention of VTE is of particular importance.Venous thromboembolism (VTE), manifesting mainly as deep venous thrombosis (DVT) and pulmonary embolism (PE), is a common serious complication of gynecologic surgery7. Heparin, including unfractioned heparin and low molecular weight heparin (LMWH), is the most commonly used pharmacologic agent for the prophylaxis of VTE. Many clinical trials indicate that heparin is a reliable method to decrease VTE in postoperative patients8. However, high-dose heparin is associated with a risk of perioperative bleeding, while low-dose heparin was found to be inefficient in high-risk patients with gynecologic cancer9. Mechanical prophylaxis, including passive methods such as graduated compression stockings (GCS) and active methods such as intermittent pneumatic compression (IPC), can avoid the risk of bleeding associated with heparin usage; however, these methods are insufficient in reducing VTE in high-risk patients or in critically ill patients12. Thus, more intense prophylactic strategies are required for high-risk patients.Many interventions and strategies are currently available to reduce the risk of VTE, such as preoperative pharmacologic prophylaxis and mechanical prophylaxis7. A systematical review indicates that dual prophylaxis is superior to monoprophylaxis in reducing postoperative VTE, even in high-risk patients with gynecologic cancer13. The American College of Chest Physicians has recommended dual prophylaxis for patients at high risk for postoperative VTE14. Consistent with these, in patients after gynecologic surgery for malignancies, the efficacy of mechanical prophylaxis in reducing postoperative VTE can be improved significantly when a pharmacologic prophylactic method was added15. Our previous study also showed that the combination of two mechanical prophylactic methods (GCS + IPC) was better than mechanical prophylaxis alone (GCS) in preventing VTE in high-risk patients after gynecologic surgery16. Combination prophylactic strategies have been recommended by some researchers for patients at very high risk for VTE after gynecologic surgery17.Compared with either pharmacologic or mechanical prophylaxis alone, dual prophylaxis has biological plausibility in further reducing postoperative VTE in high-risk patientsDespite many previous studies on dual prophylaxis for postoperative VTE prevention, the data obtained from gynecologic patients are still limited and there are currently very few large prospective randomized controlled studies. Furthermore, previous studies rarely stratified the risk of VTE among patients to identify the optimal strategy for patients at different levels of risk for VTE after gynecologic surgery. In this prospective multicenter randomized controlled study, we compared the efficacy and safety of GCS alone, GCS + LMWH, GCS + IPC, and GCS + IPC + LMWH in preventing VTE among patients who were stratified to have different levels of risk for VTE after gynecologic surgery.This multicenter randomized controlled trial was sponsored by Beijing Chaoyang Hospital of Capital Medical University and performed from June 2011 to December 2015 at five tertiary referral centers, including Beijing Chaoyang Hospital of Capital Medical University, Beijing Anzhen Hospital of Capital Medical University, The PLA General Hospital, Peking University People’s Hospital, and Beijing Tongren Hospital of Capital Medical University. The study was approved by the Ethics Committee of Beijing Chao-Yang Hospital, Capital Medical University (No. 10-Ke-42). All participants provided written informed consent before enrollment and the study was performed in accordance with the Helsinki II declaration.9/L) or coagulation disorders; (4) usage of anticoagulant drugs such as aspirin within 1 month; (5) bleeding tendency as revealed by coagulation indexes or previous intracranial or gastrointestinal bleeding; (6) congestive heart failure or pulmonary edema; (7) serious leg abnormalities , severe lower limb vascular atherosclerosis, lower limb ischemic vascular disease, or severe leg deformity; and (8) imperception of dorsalis pedis artery pulse.Women who would undergo pelvic surgery for gynecologic diseases at the above-mentioned centers were initially assessed for eligibility. Inclusion criteria were: (1) age >18 years; (2) carrying ≥1 risk factor for postoperative VTE (see below for VTE risk stratification); (3) not taking any prophylactic measures before enrollment; and (4) willing to sign a written informed consent form. Gynecologic diseases may be malignant or benign. Malignant diseases included malignancies of the ovary, uterine body, uterine cervix, vulva, and other parts of the pelvis. Benign diseases included uterine myoma, uterine adenomyoma, ovarian benign tumors, pelvic floor prolapsed, and others such as hydrosalpinx, fallopian tube abscess, encapsulated effusion, and mesosalpinx cyst. Exclusion criteria included: (1) preoperative thrombophlebitis or PE; (2) preoperative acute lower extremity venous thrombosis; (3) preoperative thrombocytopenia , GCS + LMWH , GCS + IPC , or GCS + IPC + LMWH . A simple randomization method was adopted for this study. Based on the calculated sample size (see below), 660 consecutive random numbers were selected from a random number table, assigned to each patient, and then divided by four. When the remainder was 0, 1, 2, and 3, the patient was allocated to groups A, B, C, and D, respectively. The patients were included sequentially according to the date of surgery.Eligible patients were randomized into four groups to receive GCS alone , and put on again on the next morning. The patients put GCS on for ~16 hours each day, during which GCS were put off every 6–8 hours to examine if there were ischemic manifestations or skin impairments.Knee-length GCS  were used as previously described16, generally ~24 hours after surgery.The Kendall Tyco SCD response compression system (USA), a 6-chamber sequential compression device, was used. The pressure started at the ankle at 45 mmHg and moved towards to the leg at 35 mmHg and the thigh at 30 mmHg. Each compression lasted 11 seconds, and the duration of relaxation was spontaneously adjusted according to the venous filling velocity. The therapy started 30 min before surgery and discontinued while the patient can ambulate23. Periumbilical subcutaneous injection of dalteparin 5000 IU was performed 12 h after surgery, once a day for 5 days25.Dalteparin  was used for pharmacologic prophylaxisVenous blood samples were collected 7 days before and 3 days after surgery to determine hemoglobin, platelets, and coagulation indexes including prothrombin time, thrombin time, activated partial blood coagulation time, fibrinogen, and D-dimer. Routine hematological and biochemical parameters and routine urine parameters were determined 1 day after surgery.26. If VTE was found or suspected, lower limb venography was performed to confirm the presence of DVT, and computed tomography pulmonary angiography (CTPA) was performed with the Brilliance iCT system  to detect if PE was present. Once DVT or PE was diagnosed or suspected, a joint consultation involving cardiovascular and respiratory clinicians was launched to decide the treatment regimen.Screening of VTE was performed within 7 days before and 3–5 days after surgery. Initially, color Doppler ultrasound imaging of lower extremities was performed by an experienced professional using an LEGIQ E9 color Doppler system with the probe frequency set at 8.4–9 MHz28. All adverse events reported by participants were recorded by investigators at various centers, and the principal investigator judged whether to discontinue the treatment or adopt clinical interventions. For bleeding events, the location and amount of bleeding, the amount of transfusion, and the transfused blood components were recorded. The amount of bleeding was estimated as previously described29.Safety evaluation was performed as previously describedBleeding events were classified as major and minor hemorrhagic events. Major hemorrhagic events included life-threatening bleeding, bleeding occurring in major organs or locations, non-surgical site bleeding associated with a decrease in hemoglobin level by ≥20 g/L or requiring transfusion of two or more units of whole blood, surgical site bleeding requiring further interventions such as a second open or endoscopic surgery and vaginal compression therapy, and surgical site bleeding resulting in instable hemodynamics or a decrease in hemoglobin level by ≥20 g/L. Once major bleeding was found, LMWH would be discontinued immediately.Minor bleeding was defined as all other bleeding events that did not fall under the categories of major bleeding, such as surgical field bleeding, injection site bruising, drainage port bleeding, and vaginal stump bleeding. For minor superficial bleeding, such as injection site bruising and drainage port bleeding, LMWH was not discontinued. For other minor bleeding events including deep bleeding and wound hematoma, LMWH was discontinued.9/L or <50% of the baseline platelet count occurred in patients on heparin therapy, with other causes excluded30. Once heparin-induced thrombocytopenia occurred, LMWH would be discontinued immediately.Heparin-induced thrombocytopenia was diagnosed when a platelet count ≤50 × 10Lower-limb impairments included skin lesions, ulcers, and limb necrosis.Patients who were diagnosed with or suspected of having VTE underwent a joint consultation. Once a diagnosis of PE was made, the patient was referred to the respiratory department for treatment and given LMWH anticoagulant therapy, which was gradually switched to oral warfarin to achieve a stable INR of ~2.0. After 2 to 3 weeks, the patient was discharged, continued to take oral warfarin for 6 months with INR maintained at 2.0 to 3.0, and was followed for 6 months in an outpatient manner.If a diagnosis of DVT was made, the patient was given anticoagulant therapy under the guidance of a cardiovascular clinician, which was gradually switched to oral warfarin achieve a stable INR of ~2.0. After ~2 weeks, the patient was discharged, continued to take oral warfarin for 3 months with INR maintained at 2.0 to 3.0, and was followed for 6 months in an outpatient manner.For patients without postoperative VTE, they were discharged 5 to 7 days after surgery and followed for 1 month in an outpatient manner or by telephone.20, in which we found that the incidence of VTE was 18% (17/96) in the non-prophylaxis group, 12.5% (14/112) in the GCS alone group, 6% (6/94) in the IPC alone group, 1% in the LMWH alone group, and 4.8% (5/104) in the GCS + IPC group. Based on these incidence rates, we assumed that the incidence of VTE should be <1% in the GCS + LMWH and GCS + IPC + LMWH groups. Thus, a sample size of 150 in each group can provide sufficient statistical power. Adding 10% for dropouts, a final sample size was calculated to be 660. An intention-to-treat analysis was not done.The sample size of this study was calculated according to the findings of our previous studies on patients at high risk for VTE following gynecologic surgeryt-tests between groups. Categorical data, expressed as percentages, were compared by χ2 tests if the data followed a normal distribution, and otherwise by nonparametric rank sum tests. Fisher’s exact test was adopted if the sample size in any cell was <40 or if the theoretical frequency was <5. To adjust the potential confounding factors such as operative time, unconditional Logistic regression was used to screen the risk factors for VTE. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, and a trend test for the ORs from the fitting model was performed. P-values < 0.05 were considered statistically significant.All statistical analyses were performed using SPSS 23.0 software for Windows . Numerical data, expressed as mean ± standard deviation, were compared using independent samples n = 75), refusing to participate in the study (n = 5), and estimated high risk for bleeding by surgeons (n = 11). Thus, a total of 625 patients were finally included in the study, including 411 moderate-risk patients, 133 high-risk patients, and 81 very-high-risk-patients. Figure After risk stratification, 716 patients at a moderate or higher level of risk for VTE were initially identified, of whom 91 were excluded for not meeting the inclusion/exclusion criteria  (Table P > 0.05)  was 24.9 ± 3.7 kg/mP < 0.05), although there were no significant differences between groups A and B or between groups C and D. Blood loss was significantly more in group C than in the other three groups (P < 0.05). To exclude the confounding effect of operative time and blood loss, unconditional Logistic regression analysis was performed, which revealed that the difference remained statistically significant after adjusting for operative time , but not for blood loss .With regard to surgical situation, there were no significant differences among the four groups in mode of surgery, patient-controlled analgesia, anesthetic techniques, site of infusion, or blood transfusion Table . HoweverOf the 625 patients included, no perioperative death occurred, and 32 developed DVT, including 14 in group A, 6 in group B, 8 in group C, and 4 in group D. Of all cases of DVT, only one  was proximal, involving the popliteal vein, and the rest were distal, involving the calf muscular vein , peroneal vein , and posterior tibial vein  , but not between any two other groups. Of note, the incidence of DVT was significantly lower in LMWH-treated patients (group B + group D) than in non-LMWH-treated patients (group A + group C) .The overall incidence of DVT was 5.1% (32/625). As shown in Table 2 = 42.97, P < 0.01). This finding indicates that the incidence of DVT increased with the risk level when the same intervention was given. Of note, 71.9% (23/32) of DVT cases occurred in the high-risk and very-high-risk groups.With regard to risk stratification, the moderate-risk patients had the lowest incidence of DVT , followed by the high-risk patients  and very-high-risk patients , and there was a significant difference among the three groups .The incidence of DVT in moderate-risk patients in different groups ranged from 0.9% to 3.0%, and group B had the lowest incidence , followed by group C . In high-risk patients, group D had the lowest incidence , followed by group A . In very-high-risk patients, group D had the lowest incidence , while group A had the highest incidence , and there was a significant difference in the incidence of DVT between them (χn = 2) or patient refusal (n = 4). Of 12 cases of PE, no involvement of pulmonary artery trunk occurred, and all affected pulmonary segmental arteries, including 9 cases of multiple bilateral pulmonary embolisms and 3 cases of single unilateral pulmonary embolism. No fatal PE occurred. Four (1/3) cases had apparent symptoms including chest pain (n = 2), mild chest distress (n = 1), and postoperative syncope (n = 1), all of which disappeared with rest.Of 32 cases of DVT, 26 underwent CTPA, which revealed that 12 had PE and 14 did not have; six did not undergo CTPA due to hypersensitivity to contrast medium (P < 0.05), but there was no significant difference between group A and group C. Of note, the incidence of PE was significantly lower in LMWH-treated patients (group B + group D) than in non-LMWH-treated patients (group A + group C) (P < 0.05).The overall incidence of PE was 1.9% 12/625). The incidence of PE in groups A-D was 4.4% (7/159), 0.64% (1/158), 1.96% (3/153), and 0.64% (1/156), respectively . This finding indicates that the incidence of PE increased with the risk grade when the same intervention was given. Of note, 75% (9/12) of PE cases occurred in the high-risk and very-high-risk groups.With regard to risk stratification, the moderate-risk patients had the lowest incidence of PE , followed by the high-risk patients  and very-high-risk patients , and there was a significant difference among the three groups .The incidence of PE in moderate-risk patients in different groups ranged from 0.0% to 2.0%, and groups C and D had the lowest incidence (0.0% for both). In high-risk patients, group B had the lowest incidence . In very-high-risk patients, groups B and D had the lowest incidence (0% for both), while group A had the highest incidence , and there was a significant difference in the incidence of DVT between them (χP > 0.05) (Table The rate of PE in patients with DVT showed no significant difference either among the four groups or among the different risk groups (5) Table , suggestP < 0.01). These mild bleeding events mostly occurred following 2–3 injections of LMWH. After LMWH was discontinued, bleeding stopped and surgical hemostasis was not required. A total of 27 patients discontinued LMWH, but transfusion or hemostasis was not required.Table During the follow-up period, four cases sought treatment for minor vaginal bleeding, including 1 case in group A, 1 case in group C, and 2 cases in group D. Since the two cases in group D did not develop vaginal bleeding within 7 days after surgery, their bleeding may not be caused by LMWH usage, but may be associated with poor wound healing secondary to local inflammation or suture loosening. After anti-inflammatory treatment, bleeding stopped. In addition, vaginal stump bleeding with an amount like that of menstrual bleeding occurred in a patient with PE 12 days after surgery and a patient with DVT 14 days after surgery, both of whom belonged to group A. Warfarin was discontinued in both cases until bleeding stopped, and then LMWH+ warfarin was restarted.No lower-limb impairments occurred throughout the study period.31. Directing against these predisposing factors, many prophylactic strategies have been developed to prevent postoperative VTE7. Since GCS, IPC, and LMWH are relatively commonly used strategies, the efficacy and safety of their combination in three different ways in preventing VTE after gynecologic surgery were evaluated in the present study.Three primary factors predisposing to thrombosis (Virchow’s triad) are: (1) endothelial injury; (2) stasis or turbulence of blood flow; and (3) hypercoagulability of blood7. In this study, IPC was performed using a commercial device as previously described16, while some modifications were made to the use of GCS. Since knee-length GCS are as effective as thigh-length ones, more comfortable to wear, and easier to apply19, the former was used in this study. Although the use of GCS for the entire day was advocated in many previous studies21, this is associated with an increased risk of pressure ulcer and rarely limb necrosis19. Furthermore, improperly fitted stockings may act as a tourniquet to result in venous stasis and increase the risk of VTE2. Particularly, the folding and distortion of stockings that easily occur when patients are sleeping can result in the tourniquet effect. For these reasons, we allowed our patients to put GCS off before bedtime once they can move their lower limbs , and put on again on the next morning. This greatly increased patients’ comfort and improved their compliance. As a consequence, the incidence of VTE (8.8%) in the GCS alone group was lower than that reported previously (12.5%)16, and no pressure ulcer or limb necrosis occurred throughout the study period.Both GCS and IPC are mechanical prophylactic methods, and they can increase venous return, reduce venous distention, and thus prevent venous stasis that contributes to an increased risk of VTE23. Compared to enoxaparin and nadroparin, dalteparin has a shorter plasma elimination half-life (2.8–3.8 h)33. Thus, once bleeding occurs, discontinuation of dalteparin will result in a more rapid recovery of coagulation function. Due to the combined use of dalteparin with GCS and/or IPC, dalteparin was given later (12 h vs. 2–6 h postoperatively) at a lower dose  in the present study compared to conventional administrations25. These modifications may explain why no major bleeding events occurred in this study.In this study, we used dalteparin as a pharmacological agent. As an LMWH, dalteparin has the advantages of simplicity, good anticoagulation effects, and low risk of heparin-induced thrombocytopenia7. Despite few trials on gynecologic patients, combination prophylaxis strategy has also been recommended by some researchers for this group of patients, especially those who have a high risk for VTE, such as gynecologic oncology patients after a major surgery17. In agreement with previous findings, this study indicated that combination strategies including GCS + IPC (5.2%), GCS + LMWH (3.8%), and GCS + IPC + LMWH (2.6%) were better than GCS alone (8.8%) in reducing the incidence of VTE in patients after gynecologic surgery. Thus, our study provides several additional options for combination prophylaxis of postoperative VTE in gynecologic patients.Many randomized trials performed in other fields such as urology and general surgery have demonstrated that combination prophylactic strategy is superior to either pharmacologic or mechanical prophylaxis alone in reducing the incidence of postoperative VTE8; however, high-dose LMWH may cause perioperative bleeding while low-dose LMWH has insufficient efficacy in high-risk patients9. The combination of mechanical prophylaxis with LMWH optimized for dosage and timing  may overcome these problems. In fact, although LMWH-containing strategies were associated with significantly more bleeding events, all of them were mild and manageable.A remarkable finding of this study is that LMWH-containing strategies are significantly better than non-LMWH-containing ones in the prophylaxis of postoperative VTE in gynecologic patients. Compared to the combination of two mechanical methods (GCS + IPC), the strategy combining both mechanical and pharmacologic prophylaxis (GCS + LMWH or GCS + IPC + LMWH) exhibited better efficacy. LMWH has been demonstrated to be reliable in decreasing VTE in postoperative patients18. Based on these risk factors, individual risk assessment can be performed to guide the degree of prophylaxis. This study showed that the incidence of both DVT and PE increased with the risk level even in patients receiving the same intervention, further suggesting the importance of risk stratification to guide the administration of different interventions to patients at different levels of risk. Since the majority of DVT and PE cases were present in the high-risk and very-high-risk groups, the emphasis of prophylaxis should be placed on this group of patients.Many risk factors for VTE have currently been identified7. Although all four strategies were effective in the moderate-risk patients, their efficacy still differed. Based on their efficacy, GCS + LMWH and GCS + IPC are recommended for this group of patients although all four strategies are acceptable. When patients are not suitable for the use of anticoagulants, GCS + IPC is recommended to avoid the risk of increased bleeding. For high-risk patients, both LMWH-containing strategies resulted in an acceptable incidence of DVT. From a health-economics point of view, GCS + LMWH is recommended. GCS + IPC + LMWH was associated with the lowest incidence of DVT in the very-high-risk group. Therefore, this strategy is recommended for very-high-risk patients. Of note, GCS alone are not recommended for both high-risk and very-high-risk patients due to its poor preventive effects, which is consistent with the previous finding34.An ideal prophylaxis regimen should be tailed to the patient’s thromboembolic risk, thus allowing clinicians to maximize the benefits and minimize the harms35. Future studies with larger sample size and longer follow-up periods are needed to carefully address these issues.Despite some promising results, there are several limitations to our study. First, the relatively small number of high-risk and very-high-risk patients was underpowered to show statistical differences between groups. In fact, although GCS + LMWH and GCS + IPC exhibited better efficacy than GCS alone in high-risk and very-high-risk patients, no significant difference was observed. Second, 6 of 32 patients with DVT did not undergo CTPA, and this might have resulted in the underestimation of the incidence of PE. Finally, the patients without DVT were followed for 4 weeks in this study; however, it has been reported that DVT may develop 7–12 weeks after surgeryTo conclude, this study shows that combination prophylaxis, especially LMWH-containing strategies, is superior to monoprophylaxis in reducing the incidence of VTE after gynecologic surgery. Given the incidence of both DVT and PE increases with the risk level even in patients receiving interventions, risk-stratified prophylactic strategies should be implemented in patients undergoing gynecologic surgery, with LMWH-containing strategies being recommended for high-risk and very-high-risk patients.


Pulmonary arterial hypertension (PAH) is a rare complication of dasatinib that was approved as a first-line therapy for chronic myelocytic leukemia (CML). A 24-year-old man presenting dyspnea at rest and leg edema was admitted to our hospital. He had been diagnosed with CML and prescribed dasatinib for 4 years. Chest X-ray showed significant bilateral pleural effusion and heart enlargement. Echocardiography revealed interventricular septal compression and elevated peak tricuspid regurgitation pressure gradient of 66.7 mmHg indicating severe pulmonary hypertension. After the other specific diseases to provoke PAH were excluded, he was diagnosed with dasatinib-induced PAH. Despite discontinuation of dasatinib and intravenous administration of diuretic for two weeks, World Health Organization (WHO) functional class was still II and mean pulmonary arterial pressure (PAP) was high at 37 mmHg. Therefore, we administered sildenafil and bosentan together as an upfront combination therapy three weeks after dasatinib discontinuation. Six months later, his symptoms improved to WHO functional class I and mean PAP was decreased to 31 mmHg. Although PAH is a rare complication of dasatinib, symptomatic patients prescribed with dasatinib should have an echocardiogram for PAH screening. Moreover, the upfront combination therapy would be a useful option for symptomatic patients after discontinuation of dasatinib. Pulmonary hypertension (PH) is defined as an elevation in mean pulmonary arterial pressure (PAP) of 25 mmHg or more at rest. Pulmonary arterial hypertension (PAH) is a subgroup of PH and characterized by remodeling of pulmonary vasculature, resulting in increased pulmonary vascular resistance and PAP. It is reported that some drugs including dasatinib cause PAH .Dasatinib, a second generated tyrosine kinase inhibitor (TKI), is widely used as a first-line therapy for chronic myelocytic leukemia (CML). Among the complications of dasatinib, PAH is a rare but severe complication. To treat dasatinib-induced PAH, some cases have demonstrated the effectiveness of pulmonary vasodilators . HoweverA 24-year-old man with no past history of cardiopulmonary disease visited our hospital because of worsening of dyspnea at rest and leg edema. He had been diagnosed with CML seven years ago and initially treated with imatinib. Three years later, imatinib was switched to dasatinib  because of the incomplete molecular remission.On admission, he developed increased nocturnal dyspnea, leg edema, and nausea. His blood pressure was 104/71 mmHg, heart rate was regular at 103 bpm, body temperature was 37.0°C, and oxygen saturation was 91% in room air. An X-ray examination found significant bilateral pleural effusion and heart enlargement . An elecAfter the admission as day 1, dasatinib was discontinued on day 2 and switched to bosutinib on day 14 which is another TKI used as a third choice for CML. In addition, bolus injection of loop diuretic (furosemide 20 mg daily from days 2 to 5) and oral diuretic (azosemide 60 mg qd from day 3) was started.2, and pulmonary vascular resistance was 353 dyne s cm−5, respectively  functional class was still class II, and flattening of the interventricular septum and elevated TRPG (44 mmHg) was observed by the echocardiography. In right heart catheterization (RHC) on day 15, PAP was 55 (systolic)/26 (diastolic)/37 (mean) mmHg, pulmonary capillary wedge pressure was 8 mmHg, cardiac output was 6.55 l/min, cardiac index was 3.72 l/min/mectively .Since discontinuation of dasatinib and diuretic administration had an insufficient therapeutic effect to decrease dyspnea and PAP, we started the upfront combination therapy with sildenafil, a phosphodiesterase-5 inhibitor, at 20 mg tid, and bosentan, an endothelin receptor antagonist, at 62.5 mg bid on day 20. One month later, his symptom was improved to WHO functional class I, mean PAP measured by RHC on day 15 was slightly reduced to 35 mmHg, and B-type natriuretic peptide level was decreased from 785 to 36 pg/ml. Six months later, mean PAP measured by RHC was decreased to 31 mmHg . The echWe encountered a case of severe dasatinib-induced PAH that was improved by the upfront combination therapy of vasodilators using a phosphodiesterase-5 inhibitor and an endothelin receptor antagonist.Dasatinib is a second generated TKI that was recently approved as a first-line therapy for CML. Dasatinib inhibits not only the BCR-ABL oncogenic tyrosine kinase in Philadelphia chromosome-positive CML but also other tyrosine kinases. Bosutinib is also approved for salvage therapy in CML. Unlike other TKIs, bosutinib does not significantly inhibit Kit protein or platelet-derived growth factor receptor . Among tA French group reported the incidence of dasatinib-induced PAH as 0.45% (13 out of 2900 patients) among symptomatic patients . This inIt is reported that 34 out of 36 patients (94%) with dasatinib-induced PAH recovered by discontinuation of dasatinib, but 15 out of 36 patients (41.6%) did not show complete hemodynamic recovery . MoreoveThe optimal timing and combination of the vasodilator intervention are still under debate. Since SUPER-2 trial showed that 12 weeks delay of the vasodilator made the prognosis of the patients worse , we deciWe experienced a case of dasatinib-induced PAH during the treatment of CML. We should consider the upfront combination therapy for symptomatic patients with PAH after discontinuation of dasatinib. Moreover, we strongly recommend routine screening of PAH by echocardiography during dasatinib or other TKI therapy in patients with edema or plural effusion.


Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from laboratory practice. In a subsequent study, collagen peptide sequences from an approximately 78 Myr Brachylophosaurus canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analysed by the same team, here we extract collagen from bone samples of three individuals of ostrich, Struthio camelus. The resulting LC–MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated.A decade ago, reports that organic-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approximately 68 Myr  In particular, Schweitzer et al.  reportedcollagen . With excollagen . Yet in MOR 1125 ,5. Obtai [et al.  reported [et al. .(a)T. rex specimen (MOR 1125), these initial studies were supported by two main lines of supporting molecular evidence: immunological data and sequence information [In the case of the imen MOR 125, thesormation . The autormation ,9,10 or imen MOR 25, theseet al. [T. rex specimen was likely similarly contaminated with bacterial biofilm, thus explaining the morphological similarity to the blood vessels and osteocytes that they attack. In addition, the blood-cell-like iron–oxygen spheres found in the vessels were identified as an oxidized form of formerly pyritic framboids. Interestingly, similar thin linings on Haversian canals within apatite were identified by infrared and electron microprobe analysis of ossified tendon by Manning et al. [On the grounds that mineralized and non-mineralized coatings have been found extensively in the porous trabecular bone of a variety of vertebrate fossils across time, including dinosaurs, Kaye et al.  proposedg et al.  and cleag et al. .et al. [T. rex sequence data to infer that the sample was predominantly laboratory contaminants, soil bacteria and bird-like haemoglobin and collagen. They suggested that of the six peptides that Asara et al. [Bern et al.  reanalysa et al.  depositea et al. ,13, the (b)Brachylophosaurus canadensis  bone . In this study, we aimed to investigate the differences between sequences from ostrich bone collagen and those reported for both T. rex (MOR 1125) and B. canadensis (MOR 2598).Given that the only reports that appear to favour the most recent studies cannot rule out cross-contamination, we set out to test whether or not the reported set of unique collagen peptides Three modern ostrich bone specimens were sampled, two (CC1254 and CC507) from Creswell Crags Heritage Centre  and one from our own collections (UM902) purchased from Ostrich Solutions (UK). Proteome extraction was solely restricted to our standard GuHCl-based approach following decalcification . In brie(b)® 3000 Rapid Separation LC  coupled to an Orbitrap Elite  mass spectrometer  350–1500). Peptides in the sample were separated on a 75 mm × 250 µm i.d. 1.7 µM ethylene bridged hybrid (BEH) C18 analytical column  using a gradient from 92% A (0.1% formic acid in water) and 8% B (0.1% formic acid in acetonitrile) to 33% B in 44 min at a flow rate of 300 nl min−1. Peptides were automatically selected for fragmentation by data-dependent analysis; six MS/MS scans  were acquired per cycle, dynamic exclusion was employed and one repeat scan  was acquired in a 30 s repeat duration, with that precursor being excluded for the subsequent 30 s (activation: collision-induced dissociation (CID), 2+ default charge state, 2 m/z isolation width, 35 eV normalized collision energy, 0.25 Activation Q, 10.0 ms activation time).Digested samples were analysed by LC–MS/MS using an UltiMateT-rex peptide (GPP(Oxidation)GESGAVGPAGPIGSR versus GLPGESGAVGPAGPP(Oxidation)GSR, respectively). This was done by employing a method in which peptides were automatically selected for fragmentation by data-dependent analysis; and performing six MS/MS scans. However, in this instance, three MS/MS scans were low-mass accuracy CID scans and three were high-mass accuracy higher energy collisional dissociation (HCD) scans. Each precursor that was first selected for CID fragmentation was then selected for HCD fragmentation. As described above, the CID spectra were acquired in the Velos ion trap, with the same parameters as above. HCD spectra were acquired in the Orbitrap, with a mass resolution of 15 k. All other parameters were as with the Velos, except no selection is made for Activation Q.In addition to the above, one ostrich bone proteome digest was also analysed using high resolution in the MS/MS to demonstrate the ability to resolve sequence ambiguity of the homologous ostrich peptide unique (c)Peptide spectra obtained via LC–MS/MS were searched against the SwissProt database for matches to primary protein sequences using the Mascot search engine . Each search included the fixed carbamidomethyl modification of cysteine (+57.02 Da) and the variable modifications for asparagine and glutamine deamidation (+0.98 Da), serine and threonine phosphorylation (+79.99 Da) and oxidation of lysine, proline and methionine residues  to account for PTMs and diagenetic alterations . Enzyme specificity was limited to trypsin (trypsin/P) with up to two missed cleavages allowed, mass tolerances were set at 5 ppm for the precursor ions and 0.5 Da for the fragment ions; all spectra were considered as having either 2+ or 3+ precursors and the peptide ion score cut-off was set at 30 for more confident matches. Repeat searches were also carried out using Error Tolerant search parameters with only one missed cleavage and the carbamidomethyl fixed modifications, and oxidations of both lysine and arginine selected to ensure that collagen would be adequately matched. These were carried out against the SwissProt database to retain similar search conditions to those available to the original studies, despite more avian and reptilian sequences being available elsewhere.3.(a)Brachylophosaurus and Tyrannosaurus uploaded to SwissProt GSR in T. rex), where the proposed hydroxylation of the P15 is an isoleucine in chicken, which it was originally identified as in the T. rex specimen by Asara et al. [The only peptide that appears unique to the dinosaurs is the peptide at a et al. , they cl16/b2 fragment ion pair (4). Here, we demonstrate that it can be done by acquiring an appropriate HCD spectrum for the precursor in question, i.e. at m/z 789.8972, and comparing the observed fragment ion masses with those calculated for the two carboxy-terminal peptide sequences in question . Interestingly, when we download the spectrum of the unique dinosaur peptide (100407RHad062807ndzipCID.2654.2654.2) from the published Brachylophosaurus dataset and search it against SwissProt using Mascot, the only match is to the peptide sequence (GPP(Oxidation)GESGAVGPAGPIGSR; score 50/expect value 0.0067). What is most telling is the absence of the expected y ion peak at m/z 1408.7 in their own spectrum . From both the low-mass accuracy (CID) and high-mass accuracy (HCD) spectra, it is clear that the sequence identity at residues 1–3 as GPP(Oxidation)- can be readily determined, as indicated by the yion pair . Howeverquestion . The masquestion  demonstrspectrum , but domsequence . However4.(a)Clearly, it would not be possible to disprove the hypothesis that the collagen sequences produced are indeed of dinosaur origin without sequencing an intermediate species between the extinct species in question and all of the extant taxa that had been sampled in the laboratory. Unfortunately, because ostrich was used, the species most commonly agreed as being earliest diverging from the extant bird lineage , this beBrachylophosaurus sequence does differ from our ostrich sequence rather than a matching error. However, this would place Brachylophosaurus phylogenetically closer to chicken than ostrich [In our previous statement of concern  on the o ostrich , an unliet al. [Brachylophosaurus specimen. They do point out the observations of deamidation, although these will occur at high levels depending on extraction methods, likely more so in soft tissues than bone. In order to rule out cross-sample contamination, they present one peptide for ‘ostrich tubulin’ (AILVDLEPGTMDSVR) as being different from the B. canadensis (AVLVDLEPGTMDSVR), therefore reportedly attesting to the lack of contamination by ostrich and chicken proteins in the B. canadensis extractions  is present in tubulin β-3 (NP_001074329.2) and β-5 (NP_001026183.1) chains in Gallus, tubulin β-5 (KFV86939.1) chain in Struthio (tubulin β-2 is the protein from which the above peptide they referred to originates), tubulin β-6 (XP_006269797.1) and β-4a (XP_006023414.1) chains in Alligator and several other tubulin chains among many other archosaur taxa. Likewise, AILVDLEPGTMDSVR (their ‘ostrich tubulin peptide’) is a tubulin β-2 sequence found not only in Struthio (KFV82917.1), but also in β-2 (NP_001004400.1), β-4 (NP_001026769.1) and β-7 (NP_990646.1) chains in Gallus, in β-2 (XP_006263527.3) and β-4b (XP_014462111.1) chains in Alligator and several other tubulin chains among many other archosaurs. All of the above matches are to the complete sequences, with 100% identity found from default protein BLAST searches.Cleland et al.  take thid et al.  and elec(b)B. canadensis were collected in 2006, whereas the femur was reported as being ‘protected under approximately 7 m of Judith River Formation sandstones' until 2007. It is unlikely that environmental contamination did arise at this point, but it cannot be ruled out. What is far more likely, given what we have observed with our own analyses, is laboratory cross-contamination of samples, coupled with long-term handling in collection environments (museum and research). It is not appropriate to list all specimens analysed within a particular laboratory or museum environment over a set time period . If the same laboratory has produced several publications relating to modern ostrich bone analysis, due diligence should note this as part of the samples' history. For example, Asara et al. [et al. [et al. [There are three general sources of sample contamination, either (i) in the field, whether during the recovery or beforehand, (ii) during laboratory analyses or (iii) when curated and handled in museum/research collections. In consideration of the former : the pesa et al.  used mod [et al.  used mod [et al.  used mod [et al. .et al. [The possibility of cross-contamination is typically dismissed on the grounds of sequence differences, and absence in sediment and analytical blanks. However, the latter alone cannot be considered appropriate grounds for such dismissal if the fossils are contaminated at a particular stage , whereas the sequence differences are the primary focus of our study. In this regard, the authors in their original publication  only appet al. .T. rex unique peptide (GLPGESGAVGPAGPPGSR). Nonetheless, our analyses  demonstrate that their interpretation of the ostrich peptide sequence was likely incorrect, and that highly scoring peptide matches to the T. rex sequence are also observed from modern ostrich bone analysis. Phylogenetic analyses of such data , along with a range of post-translational modifications expected with bone collagen, such as oxidations , deamidations (N and Q) and even those less commonly observed, such as glucosylgalactosyl modifications (K). Despite the overall poor sequence coverage for their modern ostrich collagen digest, one of the only two α 2(I) peptides that they did report (GLPGESGAVGPAGPIGSR) was homologous to the reported nadensis , which cet al. [et al. [Arachis hypogaea (peanut) allergen appears out of place. However, in core facilities such as these, there is typically less control over previous runs without increasing costs. This is significant as we have also noticed carry-over from other samples submitted to our own core facility that are difficult to remove from LC–MS/MS of ever-increasing sensitivity. Bern et al. [In 2011, San Antonio et al.  attempte [et al.  note than et al.  pointed n et al. ) continun et al. ,19), thi5.et al. [This report makes no attempt to address what the structures proposed by Schweitzer et al.  derive fet al. ,14, theret al.  does notet al. . Hence,


Knowledge of ovine mesenchymal stromal cells (oMSCs) is currently expanding. Tissue engineering combining scaffolding with oMSCs provides promising therapies for the treatment of osteochondral diseases.The aim was to isolate and characterize oMSCs from bone marrow aspirates (oBMSCs) and to assess their usefulness for osteochondral repair using β-tricalcium phosphate (bTCP) and type I collagen (Col I) scaffolds.in vitro cartilage repair model that was assessed using a modified International Cartilage Research Society (ICRS) II scale.Cells isolated from ovine bone marrow were characterized morphologically, phenotypically, and functionally. oBMSCs were cultured with osteogenic medium on bTCP and Col I scaffolds. The resulting constructs were evaluated by histology, immunohistochemistry and electron microscopy studies. Furthermore, oBMSCs were cultured on Col I scaffolds to develop an in vitro repair model.oBMSCs presented morphology, surface marker pattern and multipotent capacities similar to those of human BMSCs. oBMSCs seeded on Col I gave rise to osteogenic neotissue. Assessment by the modified ICRS II scale revealed that fibrocartilage/hyaline cartilage was obtained in the The isolated ovine cells were demonstrated to be oBMSCs. oBMSCs cultured on Col I sponges successfully synthesized osteochondral tissue. The data suggest that oBMSCs have potential for use in preclinical models prior to human clinical studies. Articular cartilage and its supporting bone are tightly coupled, forming a connected osteochondral unit . Orthopaβ-tricalcium phosphate (bTCP) is an absorbable ceramic that has been widely used for bone reconstruction due to its bioactive and osteoconductive properties –13. On tIn most studies, scaffolds have been used in combination with cells from different sources. Mesenchymal stromal cells (MSCs) have become attractive for cartilage and bone tissue engineering  because Preclinical studies in orthopaedic research using sheep as a large animal model are becoming common , 18–24. Knowledge of ovine MSCs is recently increasing and the sheep genome sequence was recently completed , aiding To date, most cartilage/bone engineering studies developed in both animal and human models have shown heterogeneous results  and the in vitro cartilage repair model.In this work we performed an ovine bone marrow MSC (oBMSCs) characterization using surface marker expression and multipotent differentiation. The osteogenesis of oBMSCs cultured on bTCP and Col I scaffolds was tested in depth by histological and ultrastructural analyses. Moreover, the chondrogenic repair capacity of oBMSCs cultured on Col I scaffolds was evaluated using an Ovis aries) at an age of 1 to 6 years were used  . The animals were housed in the veterinary care facility under standardized conditions of humidity and temperature with a 12 h light/dark rhythm. Animals included in this research were slaughtered in accordance with the guidelines of the Experimental Surgery Unit of the Training Technology Centre from Complexo Hospitalario Universitario de A Coruña (CHUAC), Spain.This study was approved in accordance with the Ethics Committee for Animal Experimentation of the CHUAC. For all experiments mixed breed of domestic sheep (Bone marrow samples used to obtain oBMSCs were isolated from iliac crest aspirates of sheep (n = 6). Samples of femoral condyles were obtained from previously slaughtered sheep (n = 3).Costar Corning Incorporated, New York, USA) and centrifuged after clotting with culture medium  supplemented with 5% fetal bovine serum  and 1% penicillin/streptomycin  (5%FBS/DMEM). The supernatants were discarded. Under sterile conditions, the marrow clots were divided with tweezers into several parts on adherent culture plates (Costar Corning Incorporated) and grown in culture medium 20%FBS/DMEM at 37°C in a humidified atmosphere with 5% CO2.Iliac crest bone marrow aspirates were collected into centrifuge tubes , eliminating non-adherent cells and clots, and culture medium was added. The culture medium was then replaced every 3 days.After 48 hours, the plates were washed with sterile saline solution  polypropylene tubes . The antibodies used for these experiments are listed in 5 cell events per assay was acquired using a FACsCalibur flow cytometer . Data were analyzed using Cell Quest software (BD Biosciences) and the results are expressed as mean of positive percentage ± standard error.At passages 3nd-5th passages, oBMSCs from 3 animals were differentiated towards the three mesenchymal lineages: adipocyte, osteoblast and chondrocyte.At 23 cells/cm2 in a chamber slide  for histology (n = 4) and at 50x103 in a 6-well-plate (Costar Corning Incorporated) for molecular analyses (3 independent samples). Adipogenesis was induced by culturing for 21 days in human MSC (hMSC) Commercial Adipogenic Differentiation Medium (Lonza), following the manufacturer’s instructions, with 1ng/ml of Rosiglitazone (Sigma Aldrich Quimica SA) added. Osteogenesis was induced by culture for 21 days in hMSC Commercial Osteogenic Differentiation Medium (Lonza), following the manufacturer’s instructions.oBMSCs were seeded at 5x10Both adipogenic and osteogenic differentiations were compared to a control consisting of cells cultured for the same period of time in 20% FBS/DMEM. Differentiations were confirmed by staining techniques and gene expression quantification using Real Time-PCR (qPCR).5 cells) technique [Lonza) with 10 ng/ml of human transforming growth factor (TGFβ-3)  for 21 days, following the manufacturer’s instructions. Chondrogenic differentiation was compared with cell micropellets cultured for the same period of time with 20% FBS/DMEM. After 21 days, two cell aggregates were OCT  embedded and frozen for histology and three cell micropellets were frozen, without OCT, for molecular analyses.Chondrogenesis was assessed using the micropellet formation  were supplied by the Italian company, Macrobone, Euroteknika Iberia, Barcelona, Spain) was provided by the Instituto Coruñés de Implantología y Rehabilitación Oral (ICIRO) and used to test osteogenic differentiation.1000–2000 μm grain bTCP  (2 replicates) and oBMSCs (2x105) from 3 animals at the 4th passage were also cultured with 100 mg of grain 1000–2000 μm bTCP ceramic (2 replicates), both for 30 days. Osteogenic differentiation was induced by culturing the oBMSCs in hMSC Commercial Osteogenic Differentiation Medium, following the manufacturer’s instructions. In addition, each type of scaffold, without cells, was cultured in the same medium and for the same period of time to serve as a negative control.oBMSCs from 3 animals at the 3Kai Medical, Solingen, Germany). Lesions (3 mm) were made with a dental driller  with a rotor (EWL K9).Ovine cartilage was obtained from femoral condyles  and excised with a 6 mm biopsy punch  from 2 animals (4th passage) were seeded on Col I sponges for 30 min at 37°C, and the construct was then placed inside the lesion. Punches were cultured for 8 weeks using hMSC Chondrogenic Differentiation Medium with 10 ng/ml of human TGFβ-3.oBMSCs  was analyzed using molecular biology techniques.InvitrogenTM—Thermo Fisher Scientific), following the manufacturer’s protocol. RNA was assessed for quantity at 260 nm using a NanoDropTM spectrophotometer (Thermo Fisher Scientific).Isolation of total RNA from cell culture was accomplished using Trizol Reagent (TM First-Strand Synthesis System for RT-PCR (InvitrogenTM) in a Thermocycler .The RT-PCR reaction was performed from total RNA following the manufacturer’s instructions, using the SuperScript® 480 Instrument  using LightCycler 480 SYBR Green I Master (Roche), following manufacturer’s instructions.qRT-PCR analyses were performed, using the primers and conditions shown in Roche) and REL were calculated by the 2-ΔΔCt method [Data analysis was performed for triplicates using the LightCycler 480 Relative Quantification software  staining. For osteogenesis evaluation, differentiation was analyzed by Alizarin red (AR) staining, to assess the presence of calcium deposits.TM detection kit  was used.For chondrogenesis evaluation, cell aggregates were stained with hematoxylin and eosin (HE), Masson's trichrome (MT), PAS-Alcian blue (PAS-AB) and Safranin O (SO) for Col and proteoglycans (PG). Moreover, inmunostaining for Col I, Col II and aggrecan (Agg)  was perfAssessment of osteogenic constructs and chondrogenic lesion repair was performed by histology and electron microscopy analyses.Osteogenic cell and cell-free scaffolds, and chondrogenic repair models were fixed in 4% formaldehyde and embedded in paraffin.To evaluate osteogenic constructs, samples were decalcified and stained with HE, Von Kossa (VK) and AR. Immunostaining for Col I and osteocalcin (OCN) was also performed.For cartilage repair evaluation, samples were stained as described for “Chondrogenesis evaluation”. For assessment of the quality of ovine cartilage repair, the histology scoring system, ICRS II, which was designed for use in human cartilage repair evaluation, was used. Some modifications to this grading system were made for adaptation for the analysis of ovine cartilage repair. Our modified score system comprised 7 of the 14 ICRS II parameters .National Institutes of Health, Bethesda, USA). After the colour substraction of non-stained and counterstained regions, the percentage of stained area was measured and expressed as mean ± standard error. Also, NIS-elements AR 3.0 software  was used for AR quantification.Quantitative analyses of the staining and immunostaining described above were measured using ImageJ 1.48v (Servizo de Apoio á Investigación at the University of A Coruña (SAI-UDC). For scanning electron microscopy (SEM), scaffolds were first fixed in 3% glutaraldehyde in cacodylate buffer. Before further fixation in osmium tetroxide, the scaffolds were dehydrated in a graded series of ethanol. Samples were critical-point dried by flooding with liquid CO2 in a Bal-Tec CPD 030  and gold-sputtered with the coater Bal-Tec SCD 004 (BAL-TEC). Samples were observed using a scanning electron microscope, JSM 6400 . For transmission electron microscopy (TEM) scaffolds were first fixed in 3% glutaraldehyde in cacodylate buffer and dehydrated in a graded series of acetone and then fixed in osmium tetroxide. Samples were embedded in Spurr  and 2 μm sections were stained with toluidine blue to confirm the presence of cells. Then, 60-nm-thick Spurr sections were made and observed using a transmission electron microscope, Jeol JEM 1010 (JEOL). For Energy Dispersive X-ray analysis (EDX), 5-μm-thick Spurr sections were obtained and deposited on a slide for SEM. Samples were coated with a thin layer of pure graphite carbon using BAL-TEC CEA 035 equipment (BAL-TEC). Samples were subsequently analyzed using a scanning electron microscope  equipped with a chemical microanalysis system through energy dispersive x-ray OXFORD INCA ENERGY 200 .Samples for electron microscopy were processed in the GraphPad Software, Inc.), using non-parametric tests . p-values <0.05 are considered to be statistically significant. Results are expressed as the mean ± standard error.All statistical analyses were performed with GraphPad Prism . The cell-surface antigen profile of oBMSCs was analyzed using antibodies directed against human  and rat (CD45 and CD90) CD surface markers because of the restricted number of antibodies are available for sheep (only anti-sheep CD44 was used). Although oBMSCs have cell surface antigens similar to hBMSCs, most antibodies reacting with hBMSCs did not show reactivity with oBMSCs , despitep = 0.001). Adipogenic differentiation was also assessed by qRT-PCR  for AR staining.The osteogenic differentiation potential of oBMSCs was examined by determining the presence of calcification using AR stain . 21-day-Osteogenic differentiation was also assessed by qRT-PCR . The expIndicating chondrogenic differentiation, HE staining revealed the rounded morphology of the micromass cultures. Stimulated micropellets showed higher cellularity in the core than that found in non-stimulated cultures. However, both possessed extracellular matrix (ECM), although total Col , MT and Chondrogenic differentiation was also assessed by qRT-PCR . The expp-value = 0.012, respectively for AR and 6.97%±1.31% and 2.21%±0.93%; p-value = 0.024, respectively for VK)   . Immunosprotein) .Constructs of Col I sponges, analyzed by TEM, showed cells , oval- , star-  and elonThe ultrastructural analysis of bTCP ceramic showed cells  and smalTo verify that the deposits found in the extracellular space and in the cytoplasm of the cells cultured on Col I sponges were composed of calcium phosphate, an energy dispersive X-ray (EDX) study was performed. The analysis was performed at different areas of the sample preparations to ascertain its exact composition. These areas included a region of the precipitate ; insteadCell-free Col I sponges  showed bFurthermore, more volume of ECM was observed in Col I constructs  were generated in some of the replicas  because HE staining allowed the observation of neotissue formation inside the lesion .The first parameter analyzed in the adapted ICRS II scoring system, using MT staining, was “tissue morphology” . This crThe second criteria used to evaluate PG content was “matrix staining” , which wThe next two variables, analyzed by HE staining, were “cell morphology” and “chondrocyte clustering” . In the The next item studied was “surface architecture.” The worst results showed disruptions on the surface (5%); while intermediate results revealed an upper surface smoother than the lower one (50%). The best replicas showed lack of disruptions, delaminations and loosenings, scoring 80%. The final criterion analyzed, “integration with native cartilage,” ranged from 0% (worst results) to 75–90% . Finally, to score “overall assessment,” the statistical mean of the values for all 6 parameters analyzed was calculated. The worst cartilage repair score was 19%, indicating fibrous tissue; the intermediate cartilage repair scored 52%, indicating a fibrocartilage tissue; and finally, the best neotissue formed was graded in the midrange of fibrocartilage and hyaline cartilage (70%).Some replicas have been immunostained for Col I, Col II and Agg. The collagen content of the repaired tissue was absent in the worst results, whereas in the intermediate and best replicas, both ECM proteins were present . Aggrecain vitro osteogenic differentiation of oBMSCs cultured on Col sponges and bTCP ceramic was studied, and finally, the chondrogenic differentiation of oBMSCs cultured on Col sponges in an in vitro articular cartilage repair model was analyzed.Previous studies have reported the isolation of oMSCs from bone marrow , 23–25, For our study, oBMSCs were obtained from iliac crest aspirates, the most common approach described in the literature, to collect bone marrow –25, 43. et al. [et al. [et al. [in vitro higher proliferation rate and longer life span [In our study, cells with adherence to culture plastic and a typical spindle-shaped fibroblast-like morphology were isolated. As Rentsch et al. , Al Faqe [et al.  and McCa [et al.  describe [et al.  and to sife span  than theet al. [et al. [et al. [Following the International Society of Cell Therapy criteria , hMSCs het al.  observed [et al.  observed [et al. , CD90 [1 [et al. , 25 and  [et al. , 25, 44  [et al. , 25, 44. [et al. . oBMSCs  [et al.  also wer [et al. , 48.et al. [et al. [In the literature, there are some studies that did not perform any functional characterization of oBMSCs , 15, isoet al. , who jus [et al. , who jus [et al. , 44–46,  [et al. . In othe [et al. , multipl [et al. , 20, 25.et al. [et al. [et al. [To achieve adipogenesis, a human differentiation medium was initially used without success (data not shown), confirming results obtained by Mrugala et al. . Their ret al. , 16, 49.et al. , 44, 45. [et al. . These a [et al. , 28, 44. [et al. .et al. [et al. [We observed that oBMSCs were able to differentiate towards chondrocyte-like cells only when TGFβ3 was added to the chondrogenic culture medium, as previously reported by Mrugala et al. . However [et al.  did not  [et al. . The abi [et al. , 15, 24. [et al.  allowing [et al. . Histolo [et al.  and huma [et al.  cartilaget al. [in vivo model. They found that TCP alone could not serve as an effective graft in the absence of osteoprogenitor cells, while autologous bone marrow with TCP increased bone formation. El-Jawhari et al. [Applications of MSCs, especially in combination with biomaterials, have raised optimism for future therapies to repair tissues including cartilage and bone . TCP reset al.  comparedi et al.  found thi et al. . The usei et al. , 56; thein vivo bone repair using MSCs with platelet-rich plasma (PRP)[Histologically, we found osteoblasts in Col constructs, as previously described in sma (PRP). We alsosma (PRP). Moreovesma (PRP) and calcsma (PRP) in the eet al. [et al. [The EDX analysis provides a useful tool for elemental quantification in tissues, allowing the semi-quantification of the amounts of Ca and P in bone . In our et al.  in a rat [et al.  also repet al. [et al. [et al. [Scanning electron microscopy showed different cell morphologies on bTCP and Col biomaterials. Schmitt et al.  also fou [et al. . Desanti [et al.  found twIn vitro testing provides standardized and quantifiable information about cytotoxicity, cell proliferation and differentiation capacity rate more easily than in vivo testing. Moreover, it is accepted that in vitro testing is used as a first stage test to avoid the unnecessary use of animals in materials testing [in vitro bone repair models because it is not possible to maintain long-term bone cultures. testing . Howeverin vitro studies using ovine cells and different types of scaffolds. Endres et al. [et al. [in vitro formation of cartilaginous grafts using Col I hydrogels and ovine cells. They compared the phenotype of constructs from ovine chondrocytes and oBMSCs and achieved better results using the latter. However, these previous studies did not test the constructs in a native cartilage environment as we did. We created an ovine cartilage repair model and evaluated the capacity of repair using oBMSCs and Col I constructs.Several authors , 20 have [et al.  tested tin vivo osteochondral human repair. On one hand, only histological parameters were taken into account. On the other hand, in human cartilage the presence of chondrocyte clusters is associated with a pathological state, unlike ovine cartilage where it is usual to find more than one chondrocyte per lacuna  than those described in the literature . Mathema process . However process . Likewisin vitro cartilage repair model, we demonstrated that oBMSCs cultured on Col I sponges can successfully form fibrocartilage/hyaline cartilage tissue. Finally, this study suggests that oBMSCs may have potential use in osteochondral engineering.In conclusion, the results from the present study demonstrated that oMSCs have been effectively isolated from bone marrow aspirates and showed morphological, phenotypical and functional properties similar to those observed in their human counterparts. Further optimization would be needed to use Col I scaffolds as temporary matrices for cell proliferation, migration, and osteochondral differentiation of oBMSCs. Additionally, in an S1 Fig(A) Phenotypic characterization by flow cytometry of a representative population of oBMSCs for markers characteristic of MSCs and hematopoietic cells, that did not show positivity and/or reactivity with oBMSCs (B) Phenotypic characterization by flow cytometry of mononuclear cells from ovine blood, for different clones of anti-CD45 antibody.(TIF)Click here for additional data file.S2 FigA) Images of hematoxylin-eosin (H-E) staining and type II Collagen (Col II) immunostaining performed in the best, intermediate and worst replicas. B) Images of H-E, Masson’s Thricrome (MT) and Safranin O (SO) staining and Col II, Col I and Aggrecan (Agg) immunostaining, performed in a cultured biopsy of ovine cartilage.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


MALAT1) plays an important role in vascular remodeling. Down-regulation of MALAT1 can inhibit the proliferation of vascular endothelial cells and vascular smooth muscle cells, reduce cardiomyocyte apoptosis and improve left ventricular function, which is closely linked to numerous pathological processes such as coronary atherosclerotic heart disease (CAD). The aim of this study was to investigate whether polymorphisms in MALAT1 were associated with the susceptibility to CAD.Metastasis associated lung adenocarcinoma transcript 1  were genotyped using a TaqMan allelic discrimination assay.A total of 508 CAD patients and 562 age-, gender-, and ethnicity-matched controls were enrolled in this study. Four polymorphisms in P = 0.02, 0.04, and 0.02, respectively). Moreover, CGCC haplotype was associated with a decreased risk of CAD . Multivariate logistic regression analysis identified some independent risk factors for CAD, including rs619586 and rs664589. Subsequent combined analysis showed that the combined genotypes of rs619586AG/GG and rs664589CC were associated with a reduced risk of CAD .The rs619586 AG/GG genotypes and G allele were associated with a reduced risk of CAD . Stratification analyses showed that CAD patients with rs11227209 CG/GG, rs619586 AG/GG, and rs3200401 CT/TT genotypes exhibited lower levels of TCH (MALAT1 may protect against the occurrence of CAD.These findings indicate that rs619586AG/GG genotypes in The online version of this article (10.1186/s12944-018-0728-2) contains supplementary material, which is available to authorized users. Coronary atherosclerotic heart disease (CAD) is the leading cause of death globally, resulting in 9.48 million deaths in 2016 . In addiMALAT1), known as noncoding nuclear-enriched abundant transcript 2 (NEAT2), is an infrequently spliced lncRNA more than 8000 nucleotides in length [MALAT1 plays a crucial role in the initiation of various cancers, such as lung cancer [MALAT1 has been demonstrated to implicate to the balance of vascular endothelial cell [MALAT1 is highly expressed [MALAT1 can decrease cardiomyocyte apoptosis and improve left ventricular function in diabetic rats [MALAT1 appears important not only in cancer development but also in the progression of cardiovascular diseases.Long noncoding RNAs (lncRNAs) are a group of RNAs with the length of more than 200 nucleotides. Due to lack of open reading frame, they are not translated into proteins but act as important regulators in diverse biological processes, including proliferation, apoptosis, migration, invasion, and differentiation , 10. Metn length . It is ln length . Since ig cancer , bladderg cancer , and prog cancer . Currential cell . In patixpressed , and dowtic rats . These fMALAT1 may be related to the individuals’ susceptibility to CAD. To date, SNPs in MALAT1 have been investigated in colorectal cancer [MALAT1 with the risk of CAD was reported. We carried out a case-control study to analyze the association between SNPs in MALAT1 and CAD risk in a Chinese Han population.Previous studies have shown that single nucleotide polymorphisms (SNPs) in lncRNA contribute to the risk of CAD –24. We tl cancer , lung cal cancer , 27, HBVl cancer , as welll cancer . HoweverThe study population comprised individuals of Chinese Han origin examined by coronary angiography at the Yan’An Hospital Affiliated to Kunming Medical University. A total of 508 CAD patients and 562 controls were enrolled consecutively in this study between August 2011 and October 2016. CAD diagnosis was established by angiographic evidence of significant coronary stenosis (≥ 50%) in at least one major coronary artery or a history of coronary artery bypass surgery. Patients with congenital heart disease, valvular heart disease, and infectious heart disease were excluded. The controls were selected from the physical examination program during the same period at the same hospital. They were judged to be free of CAD by clinical examination and electrocardiogram. We excluded the individuals who were not Chinese Han ethnicity or had a history of heart dysfunction. All individuals enrolled lived in the southwest of China. Hypertension was defined as a systolic blood pressure ≥ 140 mmHg and/or a diastolic blood pressure ≥ 90 mmHg at least on two distinct occasions . Diabetehttps://www.ncbi.nlm.nih.gov/snp/), we selected SNPs within the region of lncRNA MALAT1 according to the criteria of minor allele frequency more than 5% in Chinese Han population. Four SNPs  in lncRNA MALAT1 were identified.Using dbSNP database  according to the manufacturer’s protocol. SNPs genotyping was analyzed using the TaqMan allelic discrimination assay on an ABI 7500 Fast Real-Time System . The probe ID for the rs11227209, rs619586, rs664589, and rs3200401 was C_32062431_10, C_1060479_10, C_1060482_20, and C_3246069_102 test. Continuous data was expressed as mean ± standard deviation (SD) and analyzed using Student’s t test. Hardy Weinberg equilibrium (HWE) was tested by using a χ 2 goodness-of-fit test. Genotype and allele associations between lncRNA MALAT1 polymorphisms and CAD risk were evaluated by calculating odds ratios (ORs) and 95% confidence intervals (CIs). The ORs were adjusted for age, gender, hypertension, and diabetes mellitus. In multiple comparisons, P value was corrected with the Bonferroni correction and significance was defined as P < 0.0125 (0.05/4). Linkage disequilibrium (LD) and haplotype analysis were performed using SHEsis software . Multivariate logistic regression analysis was performed with independent variables, such as age, gender, hypertension, diabetes mellitus, TCH, TG, HDL-C, LDL-C, and MALAT1 polymorphisms. Differences were considered statistically significant at a P value < 0.05.SPSS for Windows version 19.0 was used for statistical analysis . Categorical data was expressed as number (percentage) and analyzed using χ P > 0.05). The frequency of hypertension and diabetes mellitus in CAD group was significantly higher than controls. Plasma levels of TCH, TG, and LDL-C were significantly higher in CAD patients, whereas HDL-C expression was significantly lower in CAD patients (P <  0.001).The general characteristics of the study population are shown in Table MALAT1 among CAD patients and controls were in HWE . The lower risk of CAD was also observed in allele comparison . However, no significant association of rs11227209, rs664589, and rs3200401 with CAD risk was found. After stratification analyses according to TCH, TG, HDL-C, and LDL-C, we found that CAD patients with rs11227209 CG/GG, rs619586 AG/GG, and rs3200401 CT/TT genotypes exhibited lower levels of TCH  (Table The distributions of the four polymorphisms in lncRNA P <  0.001) , diabetes mellitus , TCH , TG , HDL-C , LDL-C , rs619586 , and rs664589 .Some variables were taken into consideration to identify if they are independent risk factors for CAD. As shown in Table P <  0.001).As the rs619586 is a protective factor and the rs664589 is a risk factor based on logistic regression analysis, we then performed combined analysis to determine whether the rs619586 and the rs664589 have combined effects on CAD risk. As shown in Table MALAT1 might have potential function. The rs619586 A allele can bind to nuclear erythroid-related factor 2 (Nrf2), and the rs11227209 C allele and rs664589 G allele can bind to transcriptional factor SP1, whereas rs619586 G, rs11227209 G, and rs664589 C allele can not. In patients with stable coronary artery disease, Nrf2/antioxidant-related element gene expression was significantly higher than controls [Nrf2 knock out mice not only had a left ventricular diastolic dysfunction, but also had an accelerated progression to heart failure and a higher mortality rate [MALAT1 with CAD risk in a Chinese population, and we found that participants with rs619586 AG/GG genotypes had a 0.66-fold decreased risk for CAD. Stratification analyses showed that CAD patients with rs11227209 CG/GG, rs619586 AG/GG, and rs3200401 CT/TT genotypes yielded lower levels of TCH. Haplotype analysis showed that CGCC haplotype was associated with a decreased risk of CAD. Multivariate logistic regression analysis identified some risk factors for CAD, including hypertension, diabetes mellitus, TCH, TG, HDL-C, LDL-C, rs619586 and rs664589. These findings suggest rs619586 AG/GG in MALAT1 being a protective factor against CAD.In silica analysis predicted that SNPs in controls . Nrf2 knity rate , 33. SP1ity rate . Based oANRIL) conferred a significantly increased risk of myocardial infarction [ANRIL rs2383206 showed an increased risk of CAD with an adjusted OR of 2.55 [ANRIL rs4977574 G allele was more likely to increase the risk of CAD [ANRIL rs2383207 being a risk factor for CAD development [H19 was more frequent in CAD patients than in controls [lncRNA9 was significantly higher among female CAD patients compared to male patients [MALAT1 with CAD. Although no evidence of association between rs664589 and CAD risk was observed in single site analysis, multivariate logistic regression analysis showed that rs664589 was a risk factor for CAD. We then performed combined analysis of rs619586 and rs664589. Interestingly, a reduced risk of CAD with the combined genotypes of rs619586AG/GG and rs664589CC was observed, suggesting that the protective effect of rs619586 is stronger than the risk polymorphism rs664589. Previous results together with our current data indicate that lncRNA-related SNPs are involved in the etiology of CAD. It is difficult to explain why the rs619586 was not registered as a significant effect locus in genome wide association studies, some possibilities should be considered, such as environmental exposure, sample size, and ethnicity.Previous studies have shown that lncRNA-related polymorphisms may contribute to the susceptibility for CAD , 23. Chefarction . Shankerk of CAD , 21. Anoelopment . Gao et patients . In thisMALAT1 influencing CAD risk is still not clear, genetic variants in lncRNA might affect its expression through altering its secondary structure and stability [MALAT1 is abnormal in heart diseases, with high levels in acute myocardial infarction patients [MALAT1 silencing promoted migratory capacity and basal sprouting but inhibited the proliferation [MALAT1 suppressed proliferation of endothelial cells and impaired vascularization [MALAT1 knockdown reduced stiffness-dependent proliferation and migration [MALAT1 reduced cardiomyocyte apoptosis and improved left ventricular function [MALAT1 on CAD risk might be explained by the disruption of Nrf2 binding site, resulting in low expression of MALAT1. Further studies are necessary to clarify the mechanism.Although the exact mechanism of rs619586 in tability . The exppatients  and low patients . In vascferation . In vivorization . In coroigration . In diabfunction . Based oAdmittedly, the present study had a few limitations. The study design is hospital-based and restricted to a Chinese Han population, and thus the selection bias cannot be avoided. Additionally, due to moderate sample size, the association study might not have enough power to obtain the real effect. In the current study, we used chi-square to test the HWE, and found that the genotype distributions were in HWE, indicating that the selected subjects can be representative of the general population. Moreover, Quanto software was used to estimate the statistical power, and we found that power is > 80% if the relative risk is set as 1.8 under a dominant genetic model. Therefore, it appears that the observed association of rs619586 AG/GG with CAD risk is unlikely to be achieved by chance.MALAT1 on the susceptibility of CAD in the Chinese Han population. Further association studies in diverse ethnicities and functional analysis are warranted to confirm our findings.This study provides the first evidence of the protective role of rs619586 AG/GG in Additional file 1:Table S1. HWE test of lncRNA MALAT1 among CAD patients and controls. (DOCX 14 kb)


TpH), the point when afforestation changes from increasing to decreasing soil pH, are species-specific, ranging from 5.5 (Pinus koraiensis) to 7.3 (Populus spp.) with a mean of 6.3. These findings indicate that afforestation can modify soil pH if tree species and initial pH are properly matched, which may potentially improve soil fertility and promote ecosystem productivity.Soil pH regulates soil biogeochemical processes and has cascading effects on terrestrial ecosystem structure and functions. Afforestation has been widely adopted to increase terrestrial carbon sequestration and enhance water and soil preservation. However, the effect of afforestation on soil pH is still poorly understood and inconclusive. Here we investigate the afforestation-caused soil pH changes with pairwise samplings from 549 afforested and 148 control plots in northern China. We find significant soil pH neutralization by afforestation—afforestation lowers pH in relatively alkaline soil but raises pH in relatively acid soil. The soil pH thresholds ( Afforestation is often used to increase terrestrial carbon sequestration and restore ecosystem services. Here, the authors show that afforestation can also neutralize soil pH by lowering pH in alkaline soil but raising pH in acid soil, thus further promoting the restoration of ecosystem functions. Recent climate and anthropogenic changes have significantly modified soil properties including soil pH6. In particular, soil acidification has been widely reported across a variety of ecosystem types and regions7. Change in soil pH may alter soil biogeochemical processes, and has cascading effects on terrestrial ecosystem structure and functions12. For instance, soil acidification caused by nitrogen deposition has led to diversity loss throughout the world9. Further soil acidification could increase leaching loss of cation nutrients, intensifying the scarcity of some nutrient elements essential for plant growth, thus reducing plant productivity10. Accordingly, the impacts of soil acidification have attracted increasing concerns from both the scientific community and the public and raised discussions on possible mitigation measures13, and it is generally agreed that the potential impact on soil pH needs to be included in the design and evaluation of many land use change projects15.Soil pH, which measures the acidity or alkalinity of soils, is associated with many soil properties such as hydrolysis equilibrium of ions16. However, it could also change soil pH18. Soil pH is determined by the balance between production and consumption of soil hydrogen ions20, which is closely associated with nutrient  cycles23. Afforestation could affect the nutrient cycles through plant uptake of exchangeable cations23, capture of acid deposition20, modification in the quality, and quantity of litter input and rhizosphere processes20, which consequently impact the generation and consumption of soil hydrogen ions and soil pH. It is commonly reported that afforestation decreases soil pH, although results widely varied among different studies and different regions24. Furthermore, limited by research scale, we still know little on the spatial heterogeneity of afforestation-induced soil pH changes, and on how factors like background soil physical and chemical properties, and species choices may influence soil pH. Large-scale comparative studies between afforested and non-afforested control sites may provide critical information to address these questions.Afforestation is one increasingly popular type of land use change projects primarily designated for wood production, soil and water conservation, increasing carbon storage and mitigating climate change25. The Three-North Shelterbelt Development Program (TNSDP), lying in North, Northwest, and Northeast China and covering an area of 4,069,000 km2 since 197826, is one of China’s earliest large-scale afforestation projects. The TNSDP program has generated important environmental and socioeconomic benefits, including reduced soil erosion and sand storms, and increased carbon sequestration28. This large-scale afforestation project also provides a rare opportunity to investigate the effects of afforestation on soil properties. In 2012−2013, we sampled across 148 sites within the TNSDP area to conduct pairwise comparisons on soil pH between planted forests and non-afforested control plots. For each site, we sampled one non-afforested control plot and several afforestation plots (see the section “Methods”) of different stand ages, resulting in a sum of 697 sampled plots  koraiensis, Larix (L.) gmelinii, Pinus (P.) sylvestris var. mongolica, Pinus (P.) tabuliformis and Populus spp., were used in these afforestation plots; and all plots were monocultures. Our results show significant soil pH neutralization by afforestation—afforestation decreases soil pH in alkaline soils but increases soil pH in acidic soils. These findings provide improved understandings on how afforestation impacts soil pH across a broad range of soil types and afforestation tree species, which is critical for developing climate change mitigation strategies and ecological sustainability plans.The primary objective of this study is therefore to investigate the effect of afforestation on soil pH across different tree species and soil pH gradient. To achieve this goal, we conducted a comparative study using samples from northern China, a broad geographical region spanning 2000 km from east to west and known for ambitious state-sponsored large-scale afforestation efforts. China is the world’s largest cultivator of forest plantations, and afforestation has contributed to approximately 90% of its forest area expansion and 49.3% of its forest carbon sink since the 1970sp = 0.076 from a paired t-test). Afforested plots had a lower soil pH than corresponding control plots in 53.7% (295/549) of the afforestation−control pairs. In 295 of these plots, the mean and median of H+ generation were 44.32 and 6.98 mole ha−1, respectively . However, the frequency distribution of soil pH values was significantly altered by afforestation . Afforestation reduced the frequencies of both the low (pH < 5) and the high (pH > 7) soil pH values, but increased the frequency of intermediate (5 < pH < 7) pH values , although on average the change of soil pH across the 549 afforestation−control pairs was not significant —and investigated the influence of afforestation for each pH group. For the pH > 7 group, afforestation reduced soil pH in 74.1% of the pairs . This group was further divided into three sub-groups according to initial soil pH, i.e. 7–8, 8–9, and >9. Significant reduction in soil pH was found in all the three sub-groups . For the group 5 < pH < 7, afforestation increased soil pH in about a half (51.7%) of the pairs. The increase in the sub-group 5–6 was modest (mean CIPs is +0.18) but significant (p < 0.001). Furthermore, for the group with initial soil pH = 6–7, afforestation-induced soil pH change was not significant (p = 0.74).In the light of these results, we then divided the 549 pairs based on the pH value of control plots, named initial soil pH hereafter, into three groups—pH > 7, 5 < pH < 7, and pH < 5  and from 7.39 to 7.21 (p < 0.05), respectively. In contrast, afforestation with P. sylvestris var. mongolica, L. gmelinii and Populus spp. had no statistically detectable effect on soil pH , followed by that of P. koraiensis (slope −0.67), Populus spp. (slope −0.47), P. tabuliformis (slope −0.25), and P. sylvestris var. mongolica (slope −0.24); the slope was −0.31 when all the five species were combined , which is the point when afforestation-induced CIP changes from positive to negative, shown as the horizontal axis intercept of the OLS curve in Fig. TpH was about 6.3 when all the species were included, with considerably species-specific variations. Specifically, Populus spp. plantations had the highest TpH (7.3), and the P. koraiensis plantations had the lowest one (TpH = 5.5). Afforestation with L. gmelinii, P. sylvestris var. mongolica, and P. tabuliformis acidified soils when the initial soil pH was >5.8, 5.9, and 6.7, respectively. In general, Populus spp. caused the largest increase of soil pH when the initial soil pH < 5.5; but L. gmelinii and P. koraiensis resulted in the largest decrease of pH value when the initial pH > 7. Furthermore, result of two-way ANOVA showed that initial soil pH, species, and their interaction all had significant effects on CIP  curve fitting to depict the relationship between CIP and initial soil pH for each of the five afforestation species. The results suggested that afforestation decreased soil pH in alkaline soils but increased it in acidic soils consistently across all the five species Fig. . The decned Fig. . We thenCIP Fig. .Fig. 4ReThe observed neutralization effect of afforestation on soil pH may have also been confounded by some other factors such as original vegetation types, stand age, soil types, climate and net primary productivity (NPP). To test the robustness of our finding, we used generalized linear models (GLM) to evaluate the effects of all the factors  on CIP , dependent on the initial soil pH value. Afforestation acidified alkaline soils and alkalinized acid soils for all the five afforestation tree species. For a given tree species, there existed a tipping point of soil pH (TpH), where afforestation changed from increasing to decreasing soil pH. Measuring pre-afforestation initial soil pH and identifying species-specific TpH are thus essential for accurately predicting and dealing with post-afforestation soil pH changes.Collectively, our results showed that afforestation tended to neutralize soil pH. An earlier meta-analysis study of site-scale observations suggested that afforestation acidified soil globally30 may play a major role in soil pH neutralization. The addition of plant residues can increase, decrease or have little effect on soil pH35, depending on initial soil pH, nitrogen in plant materials, and the proportion of cations and organic anions. The input of plant residues decreases soil pH at high initial soil pH through nitrogen nitrification in the residue30. However, at low initial soil pH, the activity of nitrification bacteria will be suppressed and mineralized nitrogen tends to be ammonified and thus increase soil pH31. In addition, the organic anion-to-acid ratio of plant residues also has a significant role in determining whether litter decomposition would increase or decrease soil pH, subject to the value of initial soil pH as well35. Moreover, root exudates  can modify rhizospheric pH to enhance nutrient uptake by plant roots36. Roots often secrete HCO3− when plants take up more anions than cations from acid soils, leading to a higher rhizospheric pH than that of bulk soils. By contrast, in calcareous soils, plants take up more cations than anions, which reduces the rhizospheric pH by releasing H+ from their roots to maintain charge balance36. The differences in the composition of plant residues and rhizospheric processes thus lead to different CIPs across different initial soil pH and species. Furthermore, these processes also vary with soil depths and hence generate the vertical differentiations of CIP.Afforestation could lead to soil pH neutralization over the long term by altering the balance between soil hydrogen ion generation and consumption during nutrient cycle. Firstly, afforestation-caused changes in litter decomposition and rhizospheric processes40. The soil−root interface of forested soils contains more water-extractable Al than bulk soils, and afforestation may increase soil Al concentrations42 through rhizospheric processes44. The ability of hydrated aluminum ions to donate and accept H+ would make it eligible to be an acid or a base, depending on pH of soil solution. Under the domination of the reaction Al3+  + 2H2O <=> AlO2− + 4 H+, soil pH reaches an equilibrium at about 5−5.5 . When soil pH is below the equilibrium value, Al(OH)nn3− condenses to Al3+ through the reaction Al(OH)3 (s) + 3H+ → Al3+ + 3H2O, thus reducing [H+] and increasing soil pH. Here, Al3+ plays the same role as base cations  do. When soil pH is above the equilibrium value, Al3+ is hydrolyzed to Al(OH)3 (s), which releases more H+ and thus decreases soil pH. At that time, aluminum ion is an acid cation. However, these equilibrium (or threshold) pH values have only been empirically estimated by few local-scale studies in places such as Sweden39 and northeastern United States45, which may limit its applications in broader geographical regions without further verifications. In particular, the semi-arid climate in northern China’s forests and their soil properties may differ from that of Skyllberg39 and Ross et al.45. Therefore, it still remains open questions whether and how Al buffers soil pH in temperate forest of northern China.Secondly, root exudates may also indirectly influence soil pH by solubilizing unavailable soil aluminum (Al), which provides major buffering capacity in acidic soils48. It has been demonstrated that afforestation can decrease soil pH through plant uptake of base cations20. However, in acid soils, base cations are relatively scanty. Plants therefore need to get cations from deeper soil through hydrological processes20. Moreover, the increases of evapotranspiration48 caused by afforestation will reduce the leaching loss of base cations47, and thus increase soil pH. However, we did not observe a consistent relationship between CIP and change in soil moisture content (SMC) 53, respectively.Soils samples were collected from northern China, in the provinces of Heilongjiang, Jilin, Liaoning, Hebei, Shanxi, Shaanxi, and the Inner Mongolia Autonomous Region. The sampling region covered most of the TNSDP area, which extends from 34.20 to 51.80°N and 106.81 to 133.31°E , were documented in this study.We established a paired afforestation-control system to evaluate the effects of afforestation on soil pH. For each site, we chose one non-afforested plot as a control plot and several afforested plots with different stand ages ). Within a site, the distance between any afforested plot to its corresponding non-afforested control plot was less than 2.5 km to minimize the variation in soil and climatic properties between the pair; and that between any two afforested plots was more than 50 m but less than 5 km. It is noteworthy that the pre-afforestation vegetation types and soil types of afforested plots were the same as that of the corresponding control plots, according to the records provided by local forestry administrations. Specifically, the original vegetation types in this study include cropland, barren land, grassland, natural forest, and desert. Data of stand afforestation age were also obtained from local forestry administrations. For each plot, we dug three replicate soil profiles to a depth of 1 m. For each profile, soils were sampled from six layers  using a cutting ring. Therefore, except for a few plots that we could not reach to 1 m in depth, we collected 18 soil samples in each plot; and 11,118 soil samples for the whole project. We also recorded the planted tree species for each afforested plot; and as a result, five major afforestation tree species, including All soil samples were air-dried to constant weights in a ventilated room, and roots and stones were removed. Samples were then gently grinded in a mortar and passed through 2-mm sieves. The pH of each sample was measured in 1:2.5 mixtures of soil and deionized water with a pH meter . Soil solutions were shaken for 30 min and then kept static for 5 min before pH measurement.Hp) for the entire soil profile was calculated from hydrogen ion concentration, [H+], of each layer weighted by its thickness:wj and Hj are the thickness and the concentration of hydrogen ions of the jth layer, respectively. Similarly, the mean concentration of hydrogen ions in a plot . However, the hydrogen ion content varies across some orders of magnitude and it is not approximately normal distribution, so logarithmic transformation is needed. Therefore, we got the average pH for each plot from a log transformation of Because each plot included three replicate profiles and each profile included samples from different soil depths, we needed to appropriately derive the value of soil pH for each plot so that it could represent the mean concentration of hydrogen ions of soil in the whole plot. The mean concentration of hydrogen ions  correction54 was used to control potential error rates in multiple comparisons. FDR correction sorts all the p values of t-tests in ascending order: p (1) <= p (2)⋯ <= p (m), and compares each p value (p(i)) with q*i/m . If p(i) <= q*i/m, the null hypothesis is rejected. Thirdly, we used one-way and two-way analysis of variance (ANOVA) to test the effect of different factors on soil pH and CIP. Those factors used in this analysis included afforestation (control vs. afforested), tree species, and initial soil pH. Fourthly, we performed ordinary least square (OLS) to examine the correlation between CIP and initial pH. Finally, we conducted GLM to synthetically evaluate the effects of all the factors  on CIP  data, MOD17A3 data set57. Data of soil type were obtained from the Harmonized World Soil Database (HWSD) v.1.258. Longitude and latitude of each plot recorded by global positioning system were used to extract the data of climate and NPP for each plot. Furthermore, partial regressions were used to evaluate the effects of stand age on CIP after controlling for initial soil pH .Note that pH is the negative logarithm of [H(1) <= p ⋯ <= p (mThe authors declare that the source data supporting the finding of this study are provided with the paper.Supplementary Information