Our study aimed to test the hypothesis that the addition of intrathecal morphine (ITM) results in reduced postoperative opioid use and enhanced postoperative analgesia in patients undergoing open liver resection using a standardized enhanced recovery after surgery (ERAS) protocol with multimodal analgesia.A retrospective analysis of 216 adult patients undergoing open liver resection between June 2010 and July 2017 at a university teaching hospital was conducted. The primary outcome was the cumulative oral morphine equivalent daily dose (oMEDD) on postoperative day (POD) 1. Secondary outcomes included postoperative pain scores, opioid related complications, and length of hospital stay. We also performed a cost analysis evaluating the economic benefits of ITM.One hundred twenty-five patients received ITM (ITM group) and 91 patients received usual care (UC group). Patient characteristics were similar between the groups. The primary outcome - cumulative oMEDD on POD1 - was significantly reduced in the ITM group. Postoperative pain scores up to 24 h post-surgery were significantly reduced in the ITM group. There was no statistically significant difference in complications or hospital stay between the two study groups. Total hospital costs were significantly higher in the ITM group.In patients undergoing open liver resection, ITM in addition to conventional multimodal analgesic strategies reduced postoperative opioid requirements and improved analgesia for 24 h after surgery, without any statistically significant differences in opioid-related complications, and length of hospital stay. Hospital costs were significantly higher in patients receiving ITM, reflective of a longer mandatory stay in intensive care.ACTRN12620000001998.Registered with the Australian New Zealand Clinical Trials Registry (ANZCTR) under  Perioperative analgesia is critical for maximising patient satisfaction and recovery outcomes in surgery. The optimal postoperative analgesic technique for patients undergoing open liver resection is controversial. Continuous thoracic epidural analgesia has been considered the cornerstone analgesic modality, however limitations of its use in this setting include risk of epidural haematoma (in the context of coagulopathy associated with postoperative hepatic insufficiency), prolonged motor block limiting mobilisation, urinary retention, and hypotension –3. EpiduSingle shot intrathecal morphine (ITM) has recently emerged as a promising alternative practice yielding better patient outcomes –8. Intran = 5) and anaesthetists (n = 6) provided perioperative care based on a standardized liver enhanced recovery after surgery programme. As part of this protocol, all patients received an opioid based patient-controlled analgesia device for postoperative analgesia.Following prospective ethics approval by the Austin Health Human Research Ethics Committee (HREC no: LNR/18/Austin/79), we conducted a retrospective analysis of adult patients who underwent major open liver resection between July 2010 and June 2017 using a standardized ERAS protocol. All patients underwent surgery at the Austin Hospital, a university hospital in Melbourne, Australia with a dedicated high volume hepato-pancreatic-biliary and liver transplant centre. Eligible patients were identified by International Statistical Classification of Diseases (ICD) codes that included the following surgical categories: i.) excision of lesion of liver, ii.) segmental resection of liver, iii.) lobectomy of liver, iv.) trisegmental resection of liver, and v.) segmental resection of liver for trauma. In order to ensure a homogeneous patient cohort, we excluded patients receiving epidural analgesia, patients undergoing laparoscopic liver resection, and patients undergoing deroofing of liver cyst or liver biopsy. We also excluded patients with a history of chronic opioid use  for 8 weeks or longer. A team of experienced high-volume surgeons  on POD 1, adjusted for the following priori chosen variables: major resection, patient age, Charlson Comorbidity Index (CCI), duration of surgery, intraoperative oMEDD use, adjunct intrathecal clonidine, adjunct intrathecal bupivacaine, intraoperative ketamine and postoperative ketamine. These factors were chosen due to their potential influence on total morphine requirements after surgery. Secondary outcomes included average and maximum pain at rest and on movement over the first 24 postoperative hours, oMEDD use and postoperative pain scores in the PACU and on postoperative days 0, 2 and 3. We measured opioid related side effects, length of hospital stay, and performed a costs analysis (including readmissions within 30 postoperative days) for both groups of patients.oMEDD amounts were calculated using the Opioid Dose Equivalence document endorsed by the Faculty of Pain Medicine, Australian and New Zealand College of Anaesthetists . Length Complications were defined as any deviation from the normal postoperative course, guided by the European Perioperative Clinical Outcome definitions . ComplicCosts related to the index hospital admission and any consequent readmission within 30 postoperative days were included. Costs related to the preoperative and perioperative course were not considered. Allocation of costs was done based on service volume, and costs were calculated using an activity-based costing methodology. Raw costing data was obtained from the hospital’s business intelligent unit, and then allocated into categories based on individual itemisation codes for costs incurred during admission. These categories included ‘intensive care unit’, ‘medical’ , ‘pharmacy’, and ‘ward’ costs. For detailed cost analysis of complication incidence and severity, cost centres were further separated into ‘allied health’ , ‘blood products’ , ‘intensive care unit’, ‘pathology’ , ‘pharmacy’ (drug dispensing), ‘radiology’ , and ‘ward’ . Only in-hospital costs were considered, with both direct and indirect costs assessed to produce a total cost for each patient. Costs are displayed as medians and interquartile ranges to more accurately reflect the economic burden placed upon healthcare providers by catering for outliers. Costs were inflated to 2018 dollars based upon the average Australian Consumer Price Index from 2010 to 2017 inclusive, as reported by the Reserve Bank of Australia . The aveData was extracted from the patient’s electronic medical records and the Hospital’s computerized laboratory results. Austin Health utilizes Cerner® electronic health records that allows comprehensive electronic data capture and access to patient health information in the perioperative setting.Preoperative data collected included gender, age, body mass index, American Society of Anesthesiologists (ASA) class, principle diagnosis, surgical procedure, segments resected, and Charlson Comorbidity Index (CCI). The ASA score provides a simple categorisation of a patient’s physiological status before surgery and is useful in predicting perioperative morbidity and mortality . The CCIIntraoperative data collected included duration of surgery, type of resection, and whether the resection was “major” or “minor”. We collected the dose of intrathecal morphine administered just prior to the commencement of the surgery, type and amount of intraoperative analgesia delivered, and types and amounts of fluids and blood products used intraoperatively.Postoperative data collected included length of stay, duration of PCA use, time to full ward diet, time to first oral opioid use, and time in the intensive care unit (ICU). We also collected data on postoperative analgesia agents and anti-emetics administered in the post-anaesthesia care unit (PACU), on postoperative day 0 (day of surgery) and postoperative days 1 to 3. The number and severity of postoperative complications, worst sedation scores and pain scores at rest and on movement in the PACU and POD 0–3 were also collected from the patient’s electronic medical records.For the primary and the key secondary outcomes, we used quantile regression modelling adjusted for the following a-priori defined covariates: major resection, patient age, Charlson Comorbidity Index, duration of surgery, intraoperative oMEDD use, adjunct intrathecal clonidine, adjunct intrathecal bupivacaine, intraoperative ketamine and postoperative ketamine. Quantile regression models were used due to the violation of the normality of residuals assumption required for standard linear regression and lack of suitable transformations to satisfy these assumptions. Quantile regression models the association between a set of input variables and specific percentiles (or quantiles) of the outcome variable and estimates differences in the quantiles of the outcome variable between standard care and ITM groups. For example, a median (50th percentile) regression of cumulative oMEDD use at 24 h on ITM use estimates the difference in the median oMEDD use at 24 h between standard care and ITM groups adjusted for the selected covariates. For each outcome, we included three quantile regression models: the 25th percentile, the 50th percentile (median), and the 75th percentile. Standard assessment of collinearity was conducted using variance inflation factors (VIF) and condition number. For all other outcomes, continuous data was summarized as medians and interquartile range (IQR) and compared using the Mann-Whitney U test. Categorical variables were summarized as counts (proportions) and compared using the chi-squared test or Fisher’s Exact test, as appropriate. Statistical analysis was performed using statistical software RStudio . Figures were constructed using Prism 6.0 GraphPad software .n = 4), laparoscopic liver resection (n = 106), deroofing of a liver cyst (n = 5), and liver biopsy whilst staging pancreatic cancer without sufficient parenchymal resection (n = 4).Three hundred thirty-five patients underwent liver resection at the Austin Hospital between July 2010 and June 2017. Two hundred sixteen patients satisfied the inclusion criteria. Of these, 91 patients received usual care  and 125 patients received ITM in addition to usual care (ITM group). Data collection was complete for 216 (100%) patients. Reason for patients not satisfying inclusion criteria included: epidural analgesia , and 7 (4:8) respectively. The principal diagnosis and indication for resection was benign in 33 (15%) patients and malignant in 183 (85%) patients. 32 (15%) patients received chemotherapy within 3 months before surgery. 153 (71%) patients underwent a ‘minor’ (defined as 3 segments or less) liver resection, and the remaining 63 (29%) patients underwent a ‘major’ (defined as 4 segments or more) liver resection. The range of procedures performed included 35 (16%) right hepatectomies, 17 (8%) left hepatectomies, 2 (1%) central hepatectomies, 137 (63%) segmental resections (3 segments or less), and 25 (12%) extended left or right hepatectomies (5 segments or more). Baseline patient characteristics between the ITM and usual care groups is summarized in Table The median (IQR) patient age was 60 51:67) years. There were 125 (58%) males and 91 (42%) females. Median (IQR) body mass index (BMI), ASA score and CCI were 26.15 (23.1:30.4) kg/m:67 yearsp = 0.04). Whilst patients in the ITM group required less morphine compared to the control group, the effect size of this difference depended on their distribution quartile. Patients in the 25th percentile required 55.7 mg less morphine ; 50th percentile 53.3 mg less morphine , and 75th percentile 13.9 mg more morphine . A boxplot of the cumulative oMEDD consumptions on postoperative day 1 is presented in Fig. The median (IQR) cumulative oMEDD use on postoperative day 1 was 126.7 (53:268) mg in the ITM group vs. 176.3 (105:270) mg in the usual care group (Patients in the ITM group reported lower pain scores at rest and on movement over the first 24 postoperative hours (see Table p = 0.001. The ITM group received less intraoperative ketamine: median (IQR) 36 mg (20:62.5) vs. 62.5 mg (38.8:89.5) in the usual care group, p = 0.003. A higher number of patients in the ITM group required intraoperative ondansetron .For patients receiving ITM, the minimum, median (IQR), and maximum dose of intrathecal morphine delivered were 150 μg, 300 μg (250:400), and 500 μg, respectively. To enhance or lengthen the duration of intrathecal analgesia, intrathecal bupivacaine and clonidine were administered with the ITM in 26 (21%) and 13 (10%) patients respectively. The median (IQR) doses of intrathecal clonidine and bupivacaine were 50 μg (30:15) and 10 mg (2:15), respectively. All patients received additional intraoperative systemic opioids . Further, there were no significant differences observed in oMEDD use on postoperative day 1 in ITM patients who were administered other analgesic adjuncts compared to those who received ITM alone.In the post anaesthesia care unit, the median (IQR) cumulative oMEDD requirement was lower in the ITM group: 0 mg (0:10.5) compared to the usual care group 6 mg (0:21), p = 0.006. No statistically significant differences in postoperative ketamine requirements were identified at other timepoints. A detailed overview of intraoperative outcomes and postoperative opioid and ketamine requirements is presented in Table The median (IQR) postoperative ketamine requirement was lower in the ITM group on postoperative day 0: 30 mg (19.5:48) vs. 64 mg (35.8:96) in the usual care group, p = 0.001. On postoperative day 0, median (IQR) pain scores at rest in the ITM group were 3 (0:5) vs. 5 (3:7) in the usual care group, p = 0.003. No statistically significant difference in pain scores at rest on postoperative days 1–3 were observed. Median (IQR) pain scores on movement on postoperative day 0 was 3 (1:5) in the ITM group vs. 4 (3:6) in the usual care group, p = 0.007. No statistically significant differences in pain scores on postoperative days 2 and 3 between the groups were identified. For patients receiving ITM, there were no significant differences in pain scores on movement or at rest between patients receiving 150-200 μg, 300 μg or 400-500 μg of ITM.In the PACU, the median (IQR) pain score at rest was 1 (0:3) in the ITM group compared to 4 (0:7) in the usual care group, p = 0.0001. Patients in the usual care group had a greater incidence of severe sedation on postoperative day 0 . A detailed overview of postoperative and complication outcomes for each study group is presented in Table P = 0.153 Kruskal-Wallis).No statistically significant differences were identified between the ITM group and usual care group in time to full ward diet, time to first oral opioid use, complications, length of stay and 30-day readmission. Patients in the ITM group had a longer median (IQR) stay in the ICU: 17 h (12:21.5) vs. 10 h (0:18), p = 0.002; Median (IQR) total hospital costs (US$) were higher in the ITM group: $11,640  vs. $10,338 , p = 0.05. A detailed overview of postoperative costs for each study group is presented in Table Median (IQR) total hospital costs for all patients were US$11,183 8458:17,331). Costs relating to blood products, medical complications, MET calls, pharmacy, radiology and ward were similar between the ITM and the usual care group. Costs relating to allied health, ICU and pathology were higher in the ITM group. The median (IQR) costs for allied health in US$ were $454 (362.7:933.8) in the ITM group vs. $366.6 (116.5:907.8) in the usual care group, 8:17,331.We performed a single-centre observational study evaluating the opioid sparing and analgesic effects of ITM on adult patients undergoing open liver. As hypothesized, for patients undergoing open liver resection using a standardized enhanced recovery after surgery protocol with multimodal analgesia, the addition of ITM resulted in less postoperative opioid use and enhanced postoperative analgesia. Further, we found no statistically significant differences in opioid-related complications, time to mobilisation, and length of hospital stay. Hospital costs were significantly higher in patients receiving ITM, reflective of a longer mandatory stay in ICU. Our findings support the use of single shot intrathecal morphine as an efficacious analgesic technique in patients undergoing open hepatic resection.Our findings of reduced oMEDD use at 24 h after surgery in the ITM group are congruent with other studies reporting that ITM reduces postoperative opioid requirements compared to conventional analgesia strategies for up to 24 h after surgery, but not beyond , 16, 17.Interestingly, postoperative pain scores shared the same temporal pattern as oMEDD use at 24 h after surgery, and pain was significantly reduced by ITM up to 24 h after surgery, but not beyond. The medical literature evaluating ITM analgesia in open liver resection, as well as in other procedures including thoracotomy and caesarian section, mirror our findings of ITM enhancing analgesia up to 24 h post operation , 17, 19.2, PaO2, PaCO2, respiratory rate and oxygen saturation measurements [With respect to other postoperative outcomes, our findings reflect the array of studies which have universally reported that ITM causes no statistically significant difference in length of stay in open liver resection compared to other analgesic modalities , 17, 19.urements . ReflectThere are several strengths and limitations of this study. To date this is the largest review of ITM use in the context of open hepatic resection. A previous study assessing ITM in open liver resection was conducted by Sakowska et al. and involved 161 participants - less than half the size of our study , 24. In This is a single-centre study performed in a high-volume hepatobiliary unit within a tertiary healthcare centre, partly limiting the external validity of our findings. However, our hospital has all the typical characteristics of many tertiary institutions’ hepatobiliary units, and the surgical and anaesthesia perioperative protocols adopted by our centre are aligned with those in many other tertiary centres. All patients were adults who underwent open hepatic resection, which also limits the generalisability of our findings to paediatric liver resections, and to adult patients undergoing other types of surgeries. Given the retrospective nature of the study, we cannot establish a causal relationship between ITM and the change in the perioperative variables we assessed.Another significant limitation of our findings is that given the retrospective nature of our study, the collection of data may have been subject to human error in the interpretation and recording of data. However, we consider this an unlikely source of error given the comprehensive cross-checks required for data entry at our institution and the use of electronic medical records. Additionally, the data collection was conducted by a clinician not involved in postoperative patient care, thereby minimising the likelihood of detection bias. Being a retrospective observational study with no possibility of randomisation or subject blinding, our study lacks in validity compared to a randomized controlled trial. Nonetheless the large sample size, in addition to the congruence of our results with the existing medical literature, lends significant strength to our study. Our findings are hypothesis generating and may provide valuable data for power calculations for future studies on evaluating the effects of ITM on perioperative oMEDD use, analgesia, and various adverse outcomes. Finally, validation of our findings with a large multi-centre RCT, similar in design to the Multicenter Australian Study of Epidural Anesthesia Trial which involved 25 hospitals in Australia and South-East Asia, can be justified .In patients undergoing open liver resection, ITM in addition to conventional multimodal analgesic strategies reduced postoperative opioid requirements and improved analgesia for 24 h after surgery, without any statistically significant differences in opioid-related complications, time to mobilisation, and length of hospital stay. Hospital costs were significantly higher in patients receiving ITM, reflective of a longer mandatory stay in ICU. Our findings support the use of single shot intrathecal morphine as an efficacious analgesic technique in patients undergoing open hepatic resection. A RCT evaluating the effects of ITM in addition to conventional multimodal analgesic is justified.


Critical limb ischemia (CLI) is the most advanced form of peripheral arterial disease (PAD) characterized by ischemic rest pain and non-healing ulcers. Currently, the standard therapy for CLI is the surgical reconstruction and endovascular therapy or limb amputation for patients with no treatment options. Neovasculogenesis induced by mesenchymal stem cells (MSCs) therapy is a promising approach to improve CLI. Owing to their angiogenic and immunomodulatory potential, MSCs are perfect candidates for the treatment of CLI. The purpose of this study was to determine and compare the in vitro and in vivo effects of allogeneic bone marrow mesenchymal stem cells (BM-MSCs) and adipose tissue mesenchymal stem cells (AT-MSCs) on CLI treatment.For the first step, BM-MSCs and AT-MSCs were isolated and characterized for the characteristic MSC phenotypes. Then, femoral artery ligation and total excision of the femoral artery were performed on C57BL/6 mice to create a CLI model. The cells were evaluated for their in vitro and in vivo biological characteristics for CLI cell therapy. In order to determine these characteristics, the following tests were performed: morphology, flow cytometry, differentiation to osteocyte and adipocyte, wound healing assay, and behavioral tests including Tarlov, Ischemia, Modified ischemia, Function and the grade of limb necrosis scores, donor cell survival assay, and histological analysis.Our cellular and functional tests indicated that during 28 days after cell transplantation, BM-MSCs had a great effect on endothelial cell migration, muscle restructure, functional improvements, and neovascularization in ischemic tissues compared with AT-MSCs and control groups.Allogeneic BM-MSC transplantation resulted in a more effective recovery from critical limb ischemia compared to AT-MSCs transplantation. In fact, BM-MSC transplantation could be considered as a promising therapy for diseases with insufficient angiogenesis including hindlimb ischemia. Critical limb ischemia (CLI) is the most severe form of peripheral artery disease (PAD) in which associates with cardiovascular events, ulceration, high risk of limb amputation, infection, gangrene, and death , 2. CLI In the last few years, investigators have used cellular therapies in order to improve blood flow to the ischemic limb via transplanting cells that have the potential to induce the formation of new blood vessels . As a maMSCs are multipotent non-hematopoietic and fibroblast-like adherent cells that can isolate from different tissue sources, such as the bone marrow, placenta, adipose tissue, dental pulp, and umbilical cord blood , 18. TheInvestigations about stem cell-based therapy for CLI focus on the use of bone marrow mesenchymal stem cells and support by clinical evidences . MesenchTherapeutic application of stem cells for the treatment of CLI does not have to be derived only from the bone marrow but also MSCs from the adipose tissue can be used . In the 2 culture flask and were maintained at 37 °C and 5% CO2. Nonadherent cells were removed after 24 h, and the flask was washed with phosphate-buffered saline . The medium was changed regularly every 3–4 days, and at approximately 70% confluence, the cells were detached using trypsin-EDTA  and transferred to new flasks; these cells were considered as passage 1  high glucose  and 10% fetal bovine serum  with 100 U/mL penicillin–streptomycin  and cultured in a 75-cm3 pieces using a sterile scalpel and placed in the tissue plate 70 mm under sterile conditions. The tissue explants were placed in a culture dish around 5 mm space between adjacent fragments. After adhesion of explants to the plate surface (10 min), growth medium containing Dulbecco’s modified Eagle’s medium (DMEM) high glucose  and 20% fetal bovine serum  with 100 U/mL penicillin–streptomycin  was added gently to the Petri dish without disturbing the explants. After 48 h, the medium was replaced with fresh medium. On reaching 80–90% confluence, the cells were subcultured after treatment with 0.05% trypsin-EDTA . The cells were incubated in a 37 °C humidified incubator with 5% CO2 and 95% air. At passage one, the culture medium was changed to DMEM containing 10% FBS. All of the culture medium replaced every 3–4 days  to remove contaminating debris. The adipose tissue was minced into 1–2 mml-ascorbic acid, 10 mM glycerol 3-phosphate, and 100 nM dexamethasone (Sigma-Aldrich). The medium was replaced 2 times a week. After 2 weeks the cultured cells were stained with Alizarin Red Solution to visualize calcium deposits. The cells were fixed in 4% paraformaldehyde (PFA) for 20 min and stained using Alizarin Red Solution for 20 min at room temperature.For differentiation to osteocyte, passage 3 BM-MSCs and passage 3 AT-MSCs were incubated to differentiate into osteoblasts in corresponding induction medium for 2 weeks. The cells were maintained in control and osteogenic media. The control medium consisted of DMEM , supplemented with 10% FBS  and 1% penicillin/streptomycin . The osteogenic medium contained DMEM, 15% FBS , 100 μM For adipocyte differentiation, passage 3 BM-MSCs and passage 3 AT-MSCs were incubated to differentiate into adipocytes in corresponding to the induction medium for 2 weeks. The cells were maintained in control and adipogenic media. The control medium consisted of DMEM , supplemented with 10% FBS  and 1%penicillin/streptomycin . The adipogenic medium contained DMEM, 15% FBS, 100 μM L-ascorbic acid, 200 μM Indomethacin, 1000 nM insulin (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich). The medium was replaced 2 times a week. After 2 weeks, the cultured cells were stained with Oil Red-O Solution to visualize lipid vacuoles. The cells were fixed in 4% paraformaldehyde (PFA) for 20 min and stained using Oil Red-O Solution for 20 min at room temperature.For the analysis of surface marker expression of BMMSCs and ATMSCs, flow cytometry was performed. The expression of surface markers was evaluated using monoclonal antibodies against mouse anti-CD44 FITC, anti-CD105 perCP, anti-CD45 FITC, and anti-CD34 PE. Both types of cells at passage 4 were washed with PBS and then harvested via incubation in 0.05% trypsin/EDTA. After centrifugation, the cell pellets were washed with PBS and resuspended in 1 ml PBS. Then, the cells were stained with the specific antibodies in darkness. After incubation for 30 min at 4 °C, the cells were washed with PBS and then analyzed by flow cytometry. The data was analyzed with Flow Jo software (version 7.6).2 for 0, 15, 18, 20, 22, and 24 h, the migration images of the cells were observed under a microscope with ×10 , the distance of cell migration was measured, and images were taken using ImageJ Analysis software.Wound-healing assay on endothelial cells, human umbilical vein endothelial cell (HUVEC) (purchased from pasteur institute of iran), was used to study the paracrine effect of BM-MSCs and AT-MSCs on endothelial cell migration potential. After 24-h FBS starvation of MSCs, the supernatants from cells were collected. The wound-healing assay was employed by scratching the monolayer HUVEC confluent cell cultures using a sterile plastic micropipette tip. The cells were washed by DMEM and PBS twice for smoothing the edges of the scratch and removing floating cells. After that, wound healing initiated by adding the collected supernatants from MSCs on HUVEC cultures in different wells of a 6-well plate. After incubating the cells at 37 °C in 5% COAll animal experiments were performed according to the guidelines approved by the Shiraz University of Medical Sciences ethics committee (1397.430).Male C57BL/6 mice, weighing 25–30 g (6–8 week-old) were purchased from the Comparative and Experimental Medicine center at Shiraz University of Medical Sciences and were selected randomly. Animals were kept in standard conditions . They were fed a standard mouse diet and given water ad libitum.For creating critical limb ischemia (CLI) model, all animals in each group were anesthetized intraperitoneal (IP) administration using ketamine 10%  and xylazine 2% . Animals were placed in dorsal recumbency. A skin incision (about 10 mm) was done along the center of the medial thigh from the abdomen towards the knee. Subcutaneous fat tissue superficial was gently pushed away to expose the femoral neurovascular bundle. The femoral artery pushed away from the femoral vein and nerve. Subsequently, the femoral artery was isolated by blunt dissection from the femoral vein at the ligation sites between the proximal caudal femoral artery and the bifurcation of the deep femoral artery and saphenous artery. Then, two sutures were passed using 6–0 silk, transected and cauterized. The incision was closed using continuous 5–0 vicryl sutures. The operation was done under a surgical microscope .n  =  12/group): (1) control group: CLI was performed and received PBS into the ischemic site; (2) BM-MSC group: CLI was performed and received BM-MSCs; and (3) AT-MSC group: CLI was performed and received AT-MSCs. To transfer the cells into the ischemic leg, intramuscularly (IM) injection of 5 × 105 BM-MSCs and AT-MSCs that resuspended in PBS as treatment groups and PBS as a control group was done at four different sites of gastrocnemius (GC) muscle after 24 h.The male mice were randomly divided into the following three groups , and then, SRY analysis was performed. The forward primer sequence was TTTATGGTGTGGTCCCGTGGTGAG, and the reverse primer sequence was TTGGAGTACAGGTGTGCAGCTCTAC.2 field size). The mean vessel count from the three fields was used. A single countable microvessel was defined as any endothelial cell or group of cells that were clearly separated from other vessels, stroma, or tumor cells without the necessity of a vessel lumen or RBC within the lumen. Areas of gross hemorrhage and necrosis were avoided. MVD counts were measured by counting CD31 IHC hot spots in three separate 400× fields.Histological analysis of the limb tissues was done at days 7, 14, 21, and 28 after cell transplantation. To confirm that the target cells induced angiogenesis in the gastrocnemius muscle, immunohistologic staining was performed with an anti-CD31 antibody , a marker for vascular endothelial cells. The mice were euthanized at predetermined times, and gastrocnemius was removed and fixed in 10% formalin. After fixation, the tissue was embedded in paraffin, and the tissue sections were prepared and mounted on slides. Then, the tissue section slides were stained with hematoxylin-eosin (H&E) and assessed by microscopy . The tissue samples were deparaffinized by xylene and ethanol, and then, antigen retrieval was performed with proteinase K treatment. After this treatment, the tissue samples were incubated with an anti-CD31 monoclonal primary antibody at 100 μL for 15 min at room temperature. After being washed with TBS, the samples were incubated with a master polymer plus HRP at room temperature for 30 min. Then, samples were washed with TBS. Chromogen solution was prepared, 1 drop of DAB Chromogen concentrate to 1 ml of DAB substrate buffer. Samples were treated with DAB and incubated at room temperature for 5 min. The samples were covered with hematoxylin for 15 s and then washed with distilled water. After that, the dehydration step was performed by alcohol solutions at room temperature for 30 s. Finally, the samples were cleared in xylene and mounted with a permanent mounting medium. Tissue sections were analyzed using the vascular hotspot technique to obtain MVD. Sections were scanned at low power to determine areas of the highest vascular density. Within this region, individual microvessels were counted in three separate random fields at high power . Data were presented as mean ± standard deviation (SD).Statistical analysis was performed using GraphPad Prism 7 Software. Results are expressed as the mean standard error of the mean. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA), with post hoc testing performed with Bonferroni analysis or unpaired Figure The differentiation capacities of BM-MSCs and AT-MSCs to osteocyte and adipocyte obtained using Alizarin Red staining and Oil Red-O staining, respectively. For BM-MSCs, Alizarin Red staining showed that mineralized nodules formed in the BM-MSCs after 2 weeks under the osteogenic induction. Also Oil Red-O staining demonstrated that lipid-rich vacuoles formed after 2 weeks under the adipogenic induction Fig. a, b. Forp < 0.0001 for all time points). Additionally, at time points 15, 18, 20, 22, and 24 h post-scratch, the AT-MSC group showed a significant decrease in scratch area remaining compared to the control group . In fact, supernatants from both cell sources showed similarity in their ability to stimulate cell migration compared with the control group. For the control group, no scratch was 100% healed by the end of the 24-h experiment. The photos taken with a microscope in a time series then analyzed by ImageJ software in order to calculate the distance between gaps  through a paracrine manner. The supernatant of both cells led to the complete closure of the wound after 24 h. At time points 15, 18, 20, 22, and 24 h post-scratch, we documented a significant percentage decrease in the scratch area remaining between the BM-MSC compared to the control group  . In fact, CD31-MVD in BM-MSC-treated mice was significantly higher than the other groups. CD31-MVD did not significantly differ between AT-MSCs and control groups   to evaluate and monitor blood stream into the hindlimb during the regeneration process. However, the results of our functional, molecular, and cellular tests indicated that BM-MSC transplantation compared to AT-MSCs had better therapeutic effects on critical limb ischemia treatment. The evidences from induction of endothelial cell migration, muscle functional improvements, muscle restructure, necrosis grade, muscle regeneration, and formation of new blood vessels demonstrate that BM-MSC transplantation has a great potential to treat hindlimb ischemia. On the other hand, we observed all of the above factors that associated with improvements of neovascularization and blood supply were significantly elevated in BM-MSC-treated mice compared to AT-MSCs and control groups. According to our results obtained from cellular, molecular, and behavioral tests and also from previous studies that detected the level of angiogenic factors and cytokines, BM-MSCs compared to AT-MSCs have greater angiogenic potential and also secretes more amount of angiogenic factors such as VEGF and IL-8, which confirms the importance of selection of cell type for stem cell-based regenerative therapies in order to induce angiogenesis , 67. WitThe present study demonstrated the significant role of BM-MSCs in the treatment of limb ischemia. We observed improvements in functional recovery, and according to previous studies, BM-MSCs can promote neovascularization in ischemic tissues mainly through secretion of different growth factors, proangiogenic factors, and cytokines. In conclusion, BM-MSC transplantation could be considered as a promising therapy for diseases with insufficient angiogenesis including hindlimb ischemia.


Obtaining reliable and high fidelity next-generation sequencing (NGS) data requires to choose a suitable sequencing platform and a library preparation approach, which both have their inherent assay-specific limitations. Here, we present the results of successful adaptation of SureSelect hybridisation-based target enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is designed to work preferably with amplicon-based panels. In our study, we applied a custom NGS panel to screen a cohort of 16 unrelated patients affected by premature fusion of the cranial sutures, i.e. craniosynostosis (CS). CS occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. The approach presented here allowed us to achieve high quality NGS data and confirmed molecular diagnosis in 19% of cases, reaching the diagnostic yield similar to some of the published research reports. In conclusion, we demonstrated that an alternative enrichment strategy for library preparations can be successfully applied prior to sequencing on the Ion Torrent S5 platform. Also, we proved that the custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS. Successful implementation of targeted NGS in medical diagnostics results from several advantages. First, it generates disease-restricted data with fewer variants of uncertain significance, simplifying the analysis. Next, it provides very high coverage and read depth of selected regions, and finally, it limits the need for expensive laboratory equipment and data storage7. In order to generate reliable, high fidelity NGS data one has to choose a suitable sequencing platform and a library preparation protocol8. Different NGS platforms are known to have their specific limitations, such as underrepresentation of sequences with high guanine-cytosine (GC) content in case of Illumina or homopolymer length estimation bias in Ion Torrent semiconductor-based sequencing systems14. In addition to dissimilarities of NGS platforms and their specific inbuilt artefacts, also the sample preparation protocols differ in many aspects, including enrichment strategy. Currently, two major targeted enrichment strategies are available, i.e. PCR-based methods and hybridisation-based protocols. Although targeted PCR-based amplicon approach offers both easy workflow and shorter reaction time, requiring low DNA input at the same time, it suffers from several limitations, such as lower sequencing complexity and coverage uniformity18. In general, the problem of non-specificity in PCR-based methods often cannot be circumvented by careful primer design, as the oligonucleotides have usually very short sequence19. On the other hand, an alternative approach, i.e. hybridisation-based enrichment protocols such as SureSelect (Agilent Technologies) is available and is commonly applied on the Illumina platforms. The SureSelect approach is based on biotinylated RNA oligomers of substantially greater length (120 bp), which can bind to DNA more specifically and consequently enrich the targeted regions of the genome, avoiding repetitive or non-specific amplification. Therefore, SureSelect enrichment strategy allows for obtaining better sequencing complexity and coverage uniformity16.Routine NGS diagnostics requires high-quality sequencing data, short turnaround time and reasonable cost of the investigations. Therefore, out of the three major NGS-based diagnostic strategies, i.e. whole exome sequencing (WES), whole genome sequencing (WGS), and targeted gene panel sequencing, the final approach is ubiquitous and broadly applied in the clinical settingsTo our knowledge, SureSelect libraries have not been used so far to carry out the sequencing on the Ion Torrent S5 semiconductor-based platform. In this report, we present the first example of a successful adaptation of the hybridisation-based SureSelect enrichment protocol to the sequencing on the Ion Torrent S5 system. In addition, using a cohort of patients presenting with craniosynostosis, we emphasise the utility of targeted gene panel sequencing in the diagnostics of this aetiologically heterogeneous condition.20. Routine diagnostic screening of common craniosynostosis-associated genes  enables to establish genetic aetiology in 21%21 to 62%22, depending on the size of the study, ethnicity of the population, and range of the molecular analysis (either hot-spot screening or the entire gene sequencing). Since targeted NGS is regarded as a useful diagnostic method in identification of causative variants, especially in genetically heterogeneous diseases24, we have designed and applied a custom hybridisation-based panel (Agilent Technologies) to screen CS patients with negative results of preliminary molecular screening .Craniosynostosis (CS), premature fusion of one or more cranial sutures, occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disordersWe used an NGS targeted gene panel approach to screen 16 consecutive patients with CS in whom the result of conventional Sanger sequencing of preliminary molecular screening was negative. Distribution of prematurely fused sutures was as follows: coronal – 6/16 , metopic – 5/16, sagittal – 3/16, multiple – 2/16. 56.25% of patients from our cohort presented with the syndromic form of CS, whereas 43.75% had an isolated defect. All patients were subjected to a careful dysmorphological assessment upon which clinically recognisable craniofacial malformations and other defects were photographically documented. Additionally, diagnostic imaging, including X-rays, CT scans, or head MRI was performed. DNA was extracted from venous blood samples of index patients and their parents.On the basis of clinical reports available in medical literature and databases  we have chosen the gene and variant content and designed a hybridisation-based panel comprised of 61 genes .In each sample the estimated coverage exceeded 50 reads for over 95% of the target gene sequence see Fig. . Mean cond and 3rd toes. X-ray examination of the feet showed hypoplastic middle phalanges of all toes and the relative widening of 1st metatarsals as well as broadening of all bones forming the halluces   and predicted the significance of individual variants. During the quality control, out of 2565 called variants, 87 (3.4%) were dropped as artefacts. In three cases, we detected the variants definitely causative for the patients’ phenotypes. Patient 1 was suspected of Pfeiffer syndrome, based on the clinical assessment. His phenotype involved sagittal CS, maxillary hypoplasia, high palate, proptosis, broad halluces, and skin syndactyly of 2see Fig. . Upon NGsee Fig. . PathogeFGFR2 NM_000141.4: c.1694A > G, NP_000132.3:p.Glu565Gly   and c.3062G > A, p.Arg1021Gln   as well as partial left-sided lambdoid synostosis, marked craniofacial asymmetry, hearing impairment, scoliosis, bilateral split foot malformation with syndactyly of the remaining postaxial toes, extremely short and hypoplastic thumbs and 5see Fig. . The twoIntellectual development was normal in all three presented patients.th century, researchers have been extensively investigating the molecular background of CS25. The first causative gene for this condition was identified by Jabs et al. in 1993, who described a pathogenic variant within MSX2 in a family affected by autosomal dominant CS26. In the next four years, a few novel genes – FGFR1, FGFR2, FGFR3, TWIST1 – have been linked to premature fusion of the cranial sutures31. Recently, the development of high-throughput NGS-based strategies allowed for unravelling of the molecular basis of the condition at an unprecedented scale, as it happened for newly described variants, e.g. within ERF, SMAD6, and TCF1234. However, in a significant percentage of cases, pathogenesis of the craniosynostosis is still unknown or only partially understood36. Thus, there is an unquestionable need of further research by means of WES or WGS to find novel genes or non-coding variants responsible for the development of CS in humans. In the diagnostic setting, however NGS-based panel approach appears to be a sufficient solution for mutational screening of all known causative genes or variants.Craniosynostoses encompass a group of distinct, clinically variable phenotypes. Since the first dysmorphological description of the disease by Wheaton in late 1939. Interestingly, the variant detected in Patient 7, who presented with complex CS, macrocephaly, prominent forehead, flat face, proptosis, and broad halluces may not only give rise to Pfeiffer or Crouzon syndromes with normal intellectual development, but also to a more severe cloverleaf skull phenotype with an early demise40. A broad phenotypic spectrum resulting from the same pathogenic variant suggests a possibility of other yet unidentified genetic or environmental modifiers, as indicated by Oldridge and colleagues41.Here, we proved that a custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS. We investigated 16 unrelated patients and provided a diagnosis at a molecular level for 3 (19%) of them, demonstrating the high coverage and high quality of the sequencing data at the same time. In Patient 1 and 7, the pathogenic variants were previously described in individuals affected either by Crouzon or Pfeiffer syndromes. Clinical evaluation of our patients was consistent with the diagnosis of Pfeiffer syndromeRECQL4 variants, both described as causative for Rothmund-Thomson syndrome (RTS) in osteosarcoma association study42. Both variants are very likely to occur in patient 15 in trans orientation, as they were identified in heterozygous state in two different probands42. Additionally, both p.Arg1021Pro and p.Pro103Leu RECQL4 alterations represent rare or extremely rare founder mutations already described in GnomAD/ExAC database. The variant p.Arg1021Pro was annotated in heterozygosity in 2 out of almost 280 thousand control alleles, while the variant p.Pro103Leu in 168 out of about 280 thousand alleles, clearly suggesting that those two SNVs do not represent a common haplotype. Importantly, none of the variants was found in homozygosity in a control healthy population, additionally indicating high likelihood of their pathogenicity. Unfortunately, we were unable to check the parental status for the RECQL4 mutations, as the parents disagree to undergo genetic testing. Interestingly, mutations within RECQL4 gene are linked to three distinct autosomal recessive conditions with overlapping phenotypes, i.e. Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndrome (BGS), but only the last disorder comprises hearing loss in its clinical spectrum45. Considering lack of poikiloderma and the presence of hypoacusis in Patient 15, our final diagnosis was BGS. With clinical and molecular data presented here, we have broadened the phenotypic spectrum of previously reported RECQL4 alterations that may give rise to either RTS or BGS phenotype.Patient 15 carried two pathogenic 21. Importantly, we analysed only the patients with negative results of Sanger sequencing for TWIST1 alterations and the most common pathogenic hot-spot variants of FGFR1, FGFR2, FGFR3. Consequently, our diagnostic score was significantly lower than e.g. 62% reported by Paumard-Hernández et al., who did not perform any molecular prescreening in the analysed individuals22.Although we confirmed the molecular diagnosis only in 3 out of 16 probands, the diagnostic yield of 19% is equal to some of the published research reports (21%)Our study, in which a cohort of CS patients was utilised as an example, demonstrated the usefulness of targeted gene panel sequencing in the diagnostics of complex, genetically heterogeneous conditions. To our knowledge, we were the first to adapt SureSelect hybridisation-based enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is intended to work preferably with amplicon-based panels (f.e. AmpliSeq® Thermo Fisher Scientific). Agilent hybridisation technology has not been previously used on Ion Torrent S5 equipment, hence the total cost of the analysis is higher than the standard ThermoFisher Scientific procedure. This is due to the fact that our experiment was focused on obtaining the most optimal quality of sequencing data and not on the cost reduction. The estimated price for the analysis was about 1.7 times higher per sample compared to the standard procedure, but optimisation of cost is certainly achievable. Although SureSelect hybridisation-based protocol is about 1.5 times more time consuming and represents a costlier alternative in comparison to amplicon-based approach, it provides several advantages, especially in the diagnostic setting, such as reduction of PCR-related edge artefacts, better and more exact matching of hybridisation probes. Consequently, it allows for obtaining higher specificity of the amplified region.FGFR2, which was not possible in amplicon-based protocols . Moreover, we were able to include all of the listed genes, which was impossible using even an advanced made-to order option in Ion AmpliSeq Designer tool (Thermo Fisher Scientific).With the approach presented here, we achieved high molarity of both pooled libraries (756 and 525 pmol/l) and exceeded coverage of 50 reads in each sample for over 95% of the target gene sequences (173.794 kb), as well as an average coverage of 240 reads per gene across all samples. Importantly, we obtained full coverage for all of the exons within In conclusion, we successfully adapted hybridisation-based SureSelect enrichment protocol for the Ion Torrent S5 platform, demonstrating that an alternative enrichment strategy for library preparations can be applied prior to sequencing on the Ion Torrent S5. Additionally, we proved the efficiency and clinical utility of the designed gene panel in the genetic testing of patients affected by variable CS.th June 2017). All patients and their parents agreed to participate in this study. This research involved human participants under the age of 18 years. Hence we obtained informed consents from parents and/or legal guardians. We present information or images that could lead to the identification of study participants. Accordingly, a specific consent has also been obtained from all parents and/or legal guardians for publication of identifying information/images in an online open-access publication.All procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethics approval was granted by the Institutional Review Board of Poznan University of Medical Sciences (no 742/17 obtained on 22FGFR1 (c.755C > G p.Pro252Arg), exons 7 and 8 of FGFR2 (c.755C < G p.Ser252Trp and c.758C > G p.Pro253Arg), and exon 7 of FGFR3 (c.749C > G p.Pro250Arg) as well as we analysed the entire coding sequence of TWIST1 by means of Sanger sequencing. For NGS, we used high-molecular DNA with a range of DNA Integrity Number (DIN) 6.8 to 9.6. In the next step, Ion Shear Plus reagent (Thermo Fisher Scientific) cut each genomic DNA sample (1 μg) into fragments of 50–250 bp. To obtain approximately 130 bp peaks, we adjusted the time of incubation at 37 °C to 50 minutes (step 1). Afterwards, we ligated each sample with Ion P1 Adapter and Ion Express barcode (Thermo Fisher Scientific). The ligation was as follows: 15 minutes at 25 °C, 5 minutes at 72 °C. We used a thermal cycler without a heated lid (40 °C) (step 2). Next, we proceeded to amplification of the adapter-ligated libraries through PCR reaction. To obtain an adequate yield for subsequent capture without introducing bias or non-specific products, we performed pre-capture PCR with Herculase II Fusion DNA Polymerase (Agilent Technologies) consisted of 8 cycles (step 3). After steps 1–3 we purified each sample with the use of Agencourt AMPure XP beads (Beckman Coulter Genomics), whereas after steps 1 and 3 we assessed the quality and quantity of samples on 4200 TapeStation using D1000 ScreenTape system (Agilent Technologies). In the first measurement of the samples (step 1) the obtained concentrations were between 0.229 and 6.39 ng/µl, while in the final phase (step 3) concentrations ranged from 32.2 to 69.4 ng/µl.We extracted genomic DNA from the peripheral blood lymphocytes using the MagCore® HF16 Automated Nucleic Acid Extractor and quantified each gDNA using the Agilent Technologies TapeStation 4200 and Genomic DNA ScreenTape systems. The custom panel designed by us comprised 61 genes and 11 SNPs (see Tables We prepared the NGS libraries for Ion Torrent S5 platform using hybridised capture-based target enrichment approach (SureSelect) developed by Agilent Technologies. We performed hybridisation of 750 ng in 3.4 μl of each genomic DNA library using SureSelect Target Enrichment Reagent Kit according to manufacturer’s protocol for <3 Mb capture libraries. After 17 hours of hybridisation at 65 °C, we captured the targeted molecules on streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1).We amplified purified SureSelect-enriched DNA libraries and non-template control through PCR 11 cycles) with the use of Herculase II Fusion Polymerase. Before assessing DNA quality and quantity with High Sensitivity DNA Assay on TapeStation System, we purified each sample using AMPure XP beads. Based on the evaluated concentration of SureSelect-enriched DNA libraries, we calculated the amount of each sample to be included in the pool using the following formula: volume of barcoded sample: V(f)xC(f)/nxC(i). V(f) is the final required/needed volume of the pool (20 µl), C(f) is the initial concentration of all SureSelect-enriched DNA libraries in the pool, n is the number of samples to be combined, and C(i) is the initial concentration of each barcoded sample. To avoid the presence of additional fragments in each library, we size-selected our pools by agarose gel electrophoresis using the integrated E-Gel system (Thermo Fisher Scientific), purified them using Agencourt AMPure XP beads (Beckman Coulter Genomics) and finally validated using High sensitivity DNA assay on 4200 TapeStation. The molarity of pooled libraries was 756 and 525 pmol/l, respectively  and run on automated sequencer Applied Biosystems Prism 3700 DNA Analyser.We confirmed pathogenic variants using a conventional Sanger sequencing. We designed specific primers for the amplification using Primer3 tool (see Supplementary Table coverage subcommand) against a BED file defining coding parts of canonical transcripts  running as embedded instance within Ion Torrent S5 sequencer. The resulting alignment BAMs were further processed using IonReporter 5.2 pipeline (Thermo Fisher Scientific), which incorporated variant calling. Estimation of coverage for individual genes/positions was conducted via bedtools 2.27.1 , ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and dbSNP (https://www.ncbi.nlm.nih.gov/snp) on 21/03/2018. The predictions for SIFT, PolyPhen and PhyloP (46-way) tools were retrieved from the IonReporter result files (tab-separated files). Frequency data was provided by Ensembl/VEP ; additionally GNOMAD database was queried for homozygosity/heterozygosity of individual variants . The outcomes for Combined Annotation Dependent Depletion were obtained from CADD webserver . MutationTaster results were obtained using ‘query chromosomal position’ options of the public webserver . SnpEff prediction of variant consequences was obtained using local installation of SnpEff 4.3t with default databases for hg19 reference. The effect of substitutions on splicing was assessed using ADA + RF predictors available through dbNSFP v.3.5a (dbscSNV 1.1 dataset).Available clinical significance annotation was assessed in real-time from Human Gene Mutation Database Professional .Supplementary information.Figure 1 Table 1.Supplementary 1.Supplementary 2.


Wedelia trilobata and its congener native Wedelia chinensis under mono and mixed culture. Water depth of 5 cm with any of the nutrient treatments  significantly restrained the photosynthesis, leaf nitrogen and photosynthetic nitrogen use efficiency (PNUE) of both W. trilobata and W. chinensis. While, increase in the water depth to 15 cm with low nutrient treatment (n3) reduced more of biomass of W. chinensis under mixed culture. However, relative competition interaction (RCI) was recorded positive for W. trilobata and seemingly W. trilobata benefited more from RCI under high-fluctuated water depth at 15 cm in mixed culture. Therefore, higher PNUE, more competitive ability and higher plasticity may contribute to the invasiveness of W. trilobata in wetlands.Growth behavior of different species under different habitats can be studied by comparing the production of biomass, plasticity index and relative competitive interaction. However, these functional traits of invasive species received rare consideration for determining the invasion success of invasive species at wetlands. Here, we examined the effect of water depth at 5 cm and 15 cm (static and fluctuated) with different nutrient concentrations (full-strength (n1), 1/4-strength (n2) and 1/8-strength (n3) Hoagland solution) on functional traits of invasive  Hydrological turbulence is a common stress for plants in wetland ecosystems and it inevitably imposes stress on plant communities and affects the performance of plants in various transitional terrestrial and amphibious eco-systems3. Hydrological variations within wetland often determine the pattern of plant zonation and community structure. The regime of water at wetland is characterized by the depth, length and frequency of flood5.Wetland is key habitat that regulates flow of nutrients between landscape and atmosphere because of their existence at the interface between terrestrial and aquatic zones. Wetlands are highly dynamic ecosystems in terms of hydrology and recycling of nitrogen, and considered as one of the most invaded habitats worldwide6. Simultaneous fluctuations in water and nutrient levels will affect the phenotypic plasticity and interspecific interactions among invasive and native species. High phenotypic plasticity and competitive ability are key factors of successful invasion for invasive species9. Higher phenotypic plasticity enhances adaptability of invasive species in response to changes in environment at wetlands10. Nevertheless, the competitive ability of invasive and native species varies with water availability and it may be altered by environmental variations12. These functional traits are involved directly or indirectly in the mechanism of successful plant invasion of invasive species over their co-occurring native species that experience the similar environmental selective pressures13.The fluctuations of water at different depths in the wetland ecosystem disturb the community of native species and that disturbance facilitates the primary mechanism involved in plant invasions by removing the native species15. Several studies have shown that certain plants successfully invade certain environments because of higher specific leaf area (SLA). Higher SLA can enable invasive species to acquire more resources and grow at a high growth rate than co-occurring native species17. It has also been documented in numerous studies that invasive species gain competitive advantages over co-occurring native species in introduced range through increasing SLA and leaf nutrients such as leaf nitrogen (Ln)19. Leaf nitrogen (Ln) is the most essential component for a plant, linked with higher SLA, net photosynthetic rates and photosynthetic nitrogen use efficiency and contributing to higher production of biomass23. One preceding study, Chen, et al.24 noted that invasive Alternanthera philoxeroides (Mart.) Griseb showed higher photosynthetic capacity, and produced more biomass in deep submergence. Hussner, et al.25 reported that shoot biomass, root biomass and total biomass of Myriophyllum aquaticum (vell.) Verdic were increased with increasing nutrient availability at different water levels. There are also some other conflicting views about the wetland plants that their photosynthetic and growth properties were mainly affected by availability of water at varied depth with different nutrient levels28. However, with our scope, no reports have dealt with how an invasive species and its native competitors respond to water fluctuations at different depth along with nutrient concentration levels in wetlands.Moreover, functional traits of plants that are associated with their response to environmental changes have also been central theme for predicting the fitness of plants and help plants to cope to different environmental habitatsWedelia trilobata (L.) Hitchc. (Asteraceae) and its congener, native Wedelia chinensis (Osbeck.) Merr for our studies. W. trilobata is consider as native species in the tropical region of South America30, and is found as the world’s most harmful clonal evergreen creeping invasive species31. In early 1970, W. trilobata was also found on a large-scale in southern region of China32. In China, initially it was spread rapidly from grounds to roadsides and then to agricultural fields34. W. trilobata has an ability to survive in every habitat condition. It can bear nutrient and water variations but it prefers to grow in nutrient rich environment with high amount of water35. While, W. chinensis is the native congener of W. trilobata, mostly used as medicinal plant. W. chinensis grow slowly as compared to growth rate of W. trilobata36. Since, China is among the world 10-mega biodiversity countries that play a leading role in ensuring the safety of food and agricultural production. According to environmental variation in different region it is necessary to predict the future expansion of invasive species such as W. trilobata in wetland ecosystem. With the above mentioned shortfall regarding the behavior of invasive W. trilobata in wetlands, we hypothesized that 1) W. trilobata will show better responses of their functional traits under water fluctuations at different depth than its native congener W. chinensis under both mono and mixed culture. 2) highly-fluctuated water depth along with high nutrient concentrations will facilitate the W. trilobata and make it successful invader over W. chinensis in mixed culture. This study is linked with different functional traits under different environmental conditions at wetland and, could be helpful to identify the most dominant factors that help invasive plants to become more invasive. Ultimately, the information generated through this study will help in devising the management tools to stop further expansion of this invasive plant species.Therefore, based on the responses of different functional traits to different environments, we took a successful invader A and SLA of invasive W. trilobata and its native W. chinensis (Table P < 0.001) but the interaction WD*N, WD*S and N*S were noted non-significant (P > 0.05). The interaction WD*N*S between all of them had also non-significant results on AGBm (P > 0.05). Consequently, for BGBm the interaction WD*N, N*S, and interaction between all of them (WD*N*S) had also non-significant results (P > 0.05). However, the results were noted significant for the interaction between WD and S (P < 0.01). For LA and SLA, the differences were noted significant  for all the interaction.Water depth (WD), nutrient levels (N) and species (S) under both mono and mixed culture significantly affected the AGBm, BGBm, Lis Table . In caseA and SLA of W. chinensis under both mono and mixed culture,  and 5 cm fluctuated water depth (5F) with all levels of nutrient concentrations i.e. n1, n2, n3 in mono culture as well as in mixed culture  affected AGBm, BGBm, Le, Figs. . W. trilP < 0.001) for RCI in water depth, nutrients and species and were noted significant (P < 0.01) in the interaction among water depth, nutrients, species and their interaction (Table The obtained results from the measurement exhibited the significant effect on the RCI and PI. The differences were noted highly significant P < 0.00 for RCI W. trilobata were positive when grown at the 5 cm water depth with low and high nutrient concentrations either the condition was static or fluctuated and were negative at the 15 cm water depth with low and high nutrient concentrations except at 15 cm fluctuated water depth with control nutrient concentration (15Fn1)  Table . The meaW. trilobata were higher than its native congener W. chinensis in favorable and unfavorable conditions of water depth and nutrient fluctuations  and physiological traits , respectively as compared to W. chinensis under both mono and mixed culture. W. chinensis showed no plasticity (close to zero) under mixed planting and it was noted 0.020, 0.080, 0.003, 0.010, 0.007, 0.100, and 0.060 for Pn, Ln, PNUE, LA, SLA, AGBm and BGBm, respectively . However, when considering the interaction among all factors , the results noted for Pn were non-significant (P > 0.05), but the results was noted significant  for Ln and PNUE  under both mono and mixed culture, respectively. Consequently, the values of Pn ranging from 2.35 to 8.16 μmol (CO2) m−2 s−1 were recorded for W. trilobata in mono culture, while the values of Pn ranging from 1.83 to 7.55 μmol (CO2) m−2 s−1 were noted in the mixed culture, respectively. However, for W. chinensis, the values of Pn ranged from 1.37 to 7.68 μmol (CO2) m−2 s−1 in mono culture and 1.26 to 6.40 μmol (CO2) m−2 s−1 in mixed culture, respectively  under both mono and mixed culture, respectively   Table . In somesis Fig. . The abo44. Meanwhile, high SLA is correlated with the growth development of plants and it was expected that SLA of invasive species would be higher than the native species13. The results of this study exhibited that the SLA of invasive W. trilobata was significantly higher in all water depths than the native W. chinensis  of the both the studied species to display the phenotypic plasticity as plasticity index  under high water depth promoted the growth of invasive W. trilobata and led to survive in such condition, which may partially describe the ability of W. trilobata to invade waterlogged wetland habitats. As high N supports invasiveness of the invasive species; hence avoid addition of nutrients to water bodies. Moreover, our results also helpful to understand the dynamics of species in relation to flooding in wetlands.In the present study, we have found that W. trilobata and W. chinensis were collected from the single location  of Jiangsu University, Zhenjiang, Jiangsu, P. R. China in 3rd March, 2019. About 900 ramets collected for both species and then cultured in a sand medium in a seedling tray. These seedlings irrigated daily with tapwater in order to adapt the greenhouse conditions. On 20th March, 720 ramets of both species (360 for each) were selected to conduct the following experiment. The plants were divided into two groups as mono-culture and mixed culture. Plants of both species were planted in mono  and mixed- culture (1: 1) in an internal pot (diameter: 18 cm × height: 12 cm). These pots were filled with clean washed sand and then placed to an outer pots (diameter: 28 cm × height: 35 cm).The experiment was conducted at Jiangsu University, under greenhouse conditions. The greenhouse had natural lighting with (25/18) ± 2 °C (day/night) temperature and 70% relative humidity. The ramets of th April. For water depth fluctuations, four treatments levels were established according to Sun et al.56 and Zhou et al.28: (1) static-water level at 5 cm, coded as 5S; (2) fluctuated-water level at 5 cm, fluctuating between 5 and 10 cm water depth, coded as 5F; (3) static-water level at 15 cm, coded as 15S; and (4) fluctuated-water level at 15 cm, fluctuating between 15 and 25 cm water depth, coded as 15F  were measured by analysis the images though ImageJsoftware . The specific leaf area (SLA) was calculated according to following equation:A is specific leaf area; LA is leaf area per leaf; and LDW is the leaf dry weight per leafAbout five-plants were selected from each treatment for the measurement of biomass. The measurements chosen for biomass traits analysis were above ground biomass (AGBm) and below ground biomass (BGBm). AGBm and BGBm were measured by using weighing scale. Leaf area  when grown alone, and Xb is the total dry weight of species b (invasive W. trilobata) when is grown alone. Xab is the total dry weight of species a when is grown with species b, and Xba is the total dry of species b when is grown with species a.Relative competition index (RCI) was calculated based on the total dry weight of a plant. It was the response of both invasive Plasticity index (PI) ranged from zero to one and was calculated between the two levels of more affected treatment for each growth and physiological traits. The following equation was used to measure PI:13.It can be calculated for each treatment, for each variable and for each species as the difference between the maximum value and minimum value between the two levels of each treatment divided by the maximum valuePn), leaf nitrogen (Ln) and photosynthetic nitrogen use efficiency (PNUE). Pn was measured by using a portable LI-6400XT, Lincoln, USA photosynthesis measurement system. All these data were recorded during full sunshine at 9:30–11:30 a.m. once a week during the experiment. Leaves were selected from five plants per group of treatment for the measurements.The physiological parameters selected for measurements were net photosynthetic rate (Ln) was measured using the plant chlorophyll meter, Oakoch OK-Y104 (made in China). The Ln was noted from the same leaves which were used for photosynthetic measurements. While, PNUE was calculated as the ratio of Pn values to the Ln values58.Leaf nitrogen  and graph were produced in origin pro9.A mixed model was used to evaluate the effect of the independent variables of species, water depth fluctuations, nutrient treatments and their interaction under mono and mixed culture on the dependent variables


The prolonged effects of disasters on reproductive outcomes among the survivors are less studied, and the findings are inconsistent. We examined the associations of maternal exposure to the 2008 Wenchuan earthquake years before conception with adverse birth outcomes.We included 73,493 women who delivered in 96 hospitals in 24 provinces and autonomous regions from the 2015/16 China Labor and Delivery Survey. We weighted the multivariable logistic models based on the combination of coarsened exact matching (CEM) weight and survey weight, and performed sex-stratified analysis to test whether associations of maternal earthquake exposure with adverse birth outcomes  varied by sex.P = 0.010). The sex-stratified analysis showed the association was significant among male births (aRR (95%CI): 1.40(1.12–1.75),P = 0.002),but unsignificant among female births. The sensitivity analysis reported similar findings.The bivariate models showed that the weighted incidence of each adverse birth outcome was higher in exposed group than unexposed group: stillbirth (2.00% vs. 1.33%), PTB (14.14% vs. 7.32%), LBW (10.82% vs. 5.76%), and SGA (11.32% vs. 9.52%). The multivariable models showed maternal earthquake exposure was only associated significantly with a higher risk of PTB in offspring among all births (adjusted risk ratio [aRR](95%CI):1.25(1.06–1.48), The 2008 Wenchuan earthquake exposure has a long-term effect on PTB. Maternal acute exposure to disasters could be a major monitor for long-term reproductive outcomes. More attention should be paid to the underlining reasons for disaster-related adverse birth outcomes. Adverse birth outcomes, such as low birthweight (LBW), preterm birth (PTB) and small for gestational age (SGA), are associated with numerous risk factors, including not only maternal characteristics , 2 and eMoreover, the few existing studies of prolonged effects of disasters on reproductive outcomes among the survivors reported mixed results, with one showing negative effects among women exposed to the 2001 World Trade Center disaster in Unites States  and anotThe 8.0-magnitude Wenchuan earthquake hit Wenchuan, Sichuan province on May 12, 2008, leading to 69,197 deaths, 374,176 injuries and 18,222 missing persons  among thTo bridge the gap, we evaluated the data from a national hospital-based cross-sectional study to investigate the long-term effects of the Wenchuan earthquake on birth outcomes. We hypothesized that the women exposed to the devastating earthquake and became pregnant years later would be associated with elevated risks of adverse birth outcomes in offspring. Given that male births are more vulnerable to maternal stress , we, theData in the present study were from the China Labor and Delivery Survey (CLDS), a multi-center, hospital-based cross-sectional survey throughout China to estimate the national labour and delivery information of new births between March 1, 2015, and December 31, 2016. Using a stratified multistage cluster sampling design, the CLDS selected 112 hospitals with at least 1000 childbirths per year from 25 provinces and autonomous regions across China mainland. Details on the sampling strategy of CLDS have been described elsewhere [Although the CLDS was a cross-sectional observational study, the Wenchuan earthquake generated a natural experimental setting to investigate the long-term effects of the earthquake for birth outcomes among the female survivors who became pregnancy years following the devastating earthquake , 16. In The Research Project Review Panel at the Department of Reproductive Health and Research of World Health Organization (WHO) reviewed and approved the protocol of the CLDS for technical content. The CLDS was approved by the WHO Research Ethics Review Committee and the ethics committees in all participating hospitals. Informed consent was not required, as the collected data from hospital records were anonymized and de-identified to preserve privacy for the participants.In this natural experiment, individual-level maternal exposure to the 2008 Sichuan earthquake was unmeasured in the original design and is difficult to define, as noted in the previous review , indicatThe main outcomes of interest were the outcome of delivery, gestational age, birthweight, and birthweight for gestational age. The corresponding adverse birth outcomes included stillbirth, PTB, LBW, and SGA.We defined stillbirth as the death of a baby before or during delivery after 23 weeks gestation or weighing at least 500 g at birth. We divided gestational age at birth as preterm (< 37 weeks), term (37–41 weeks) and post-term (≥42 weeks) birth. We classified birthweight into low (< 2500 g), normal (2500–3999 g) and high (≥4000 g) groups. We categorized sex-specific birth weight-for-gestational age as small (<10th percentile), appropriate (10th–90th percentile) and large (>90th percentile) categories.2), healthy weight (18.5–24.9 kg/m2), and overweight or obesity (≥25 kg/m2) [We divided maternal age into young (< 20 years), reference (20–34 years) and advanced (≥35 years) class. We categorized maternal education attainment as low (junior school or below), middle , and high (bachelor degree or above) group. We labeled the women as residents if they held the hukou in the province where they delivered, or migrants if not. According to the WHO classification of body mass index , we categorized maternal pre-pregnancy BMI as underweight (< 18.5 kg/m5 kg/m2) .Maternal medical conditions in the present study included maternal diseases , history of PTB, method of conception, parity (primipara or multipara), multiple pregnancy or singleton pregnancy, mode of delivery and sex of newborns.svy and subpop option were used to account for complex survey design.Statistical analysis was performed from January 5, 2020, to March 12, 2020. Statistical analyses were performed using Stata/SE 15.1. To obtain a national estimation of labour and delivery information, survey weight for each newborn included in CLDS was calculated using the delivery data from 2016 China Statistical Yearbook in the previous study [2 tests, and found the distribution of a few covariates was not balanced between the two groups (Table svyset command with the new weight. We reported CEM–weighted adjusted risk ratio (aRR) and 95% confidence interval (CI) using adjrr command [We first examined distributions of the exposure indicator, outcome variables, and covariates, and investigated the associations of exposure indicator with outcome variables and covariates using χps Table . To accops Table . We obta command  after weIn sensitive analyses, we restricted our analysis to singleton births, given that multiple pregnancies carry significantly higher risks of birth outcomes, including preterm birth  and stilWe drew a total of 73,493 hospital records in our primary analysis, including 34,170 female births (47.30%), 1569 multiple births (1.78%) and 10,150 pregnant migrant women (12.85%), more details of the delivery information can be found in Table P = 0.018), PTB , LBW , and SGA .The weighted incidences of adverse birth outcomes were 1.35% for stillbirth, 7.55% for PTB, 5.93% for LBW, and 9.58% for SGA in our sample Table . As showP = 0.010) among all births, and similar results were found in other subpopulation, including singleton births, all livebirths, and singleton livebirths  may be useful, but the datasets before the earthquake are not available. Third, we were unable to distinguish between the earthquake-related stress and other social stressors due to limitations in data availability. Fourth, the dose-response relationship between distance from the epicenter of the earthquake and incidence of PTB could support our findings. However, the sample size in Sichuan province is not large enough to allow us to conduct the dose-response analysis. Last, we only included secondary and tertiary hospitals in this study, which may introduce select bias and underestimate the associations. However, few pregnant women delivery in home or primary hospitals in China, and mostly due to exceptional circumstances , 37. ConMaternal exposure to the 2008 Wenchuan earthquake elevates the risk of PTB in offspring several years later after the earthquake, and only the male birth is vulnerable to the long-term effect. Maternal acute exposure to disasters could serve as a major monitor for long-term reproductive outcomes, and more research is needed to understand the underlining reasons for long-term disaster-related adverse birth outcomes.Additional file 1: Table S1. Associations of maternal exposure to Wenchuan earthquake with adverse birth outcomes among all births of local pregnant women. Table S2. Associations of maternal exposure to Wenchuan earthquake with adverse birth outcomes among all livebirths of local pregnant women.


To study the relationship between surface membrane-bound APRIL and ITP.  The peripheral blood of all subjects, 50 patients diagnosed with ITP and 25 healthy controls, was collected. Flow cytometry was used to detect the expression of membrane-bound APRIL on immune cells and platelets. ELISA was used to detect the content of soluble APRIL in plasma. P < 0.001) and chronic (P < 0.001) ITP patients than in controls. Platelet surface APRIL level was significantly enhanced in patients with complete remission after treatment (P = 0.02) but not in those with no response after treatment. Platelet surface APRIL level in ITP patients was negatively correlated with serum APRIL level .  Membrane-bound APRIL was only expressed on the surface of platelets in both ITP patients and controls. APRIL expression on the platelet surface was significantly lower in newly diagnosed ( Platelet surface APRIL may play a key immunoregulative role. Platelet surface APRIL is likely to be one source of the excessive serum APRIL in ITP patients. The effectiveness of treatment may be measured by determining the platelet surface APRIL levels in ITP patients. Primary immune thrombocytopenia (ITP) is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction and aberrant platelet production. While it may be asymptomatic for some patients, others can suffer mild, moderate, or even severe bleeding. The pathophysiology of ITP is complex but may be caused by multiple factors such as impaired megakaryopoiesis leading to reduced platelets , the proAPRIL, also known as tumor necrosis factor superfamily [TNFSF] 13, is secreted by a range of cells including myelocytes , activatSince APRIL is found to contribute to platelet production as an autocrine growth factor produced by megakaryocytes , its rolFirstly, they overlooked surface membrane-bound APRIL, since they only examined soluble APRIL in plasma. There are two forms of APRIL: soluble and surface membrane-bound . It is bKamhieh-Milz et al. , anotherIn this study, we examine the relationship between APRIL and ITP by focusing on surface membrane-bound APRIL with two objectives in mind: (1) to find out whether APRIL is expressed on the surface of immune cells and platelets in ITP patients and normal controls; (2) if yes, how surface membrane-bound APRIL respond to treatment; and (3) to explore how surface membrane-bound APRIL is related to soluble APRIL.50 ITP patients and 25 healthy controls participated in the study. The ITP patients  visited the Institute of Hematology, Chinese Academy of Medical Sciences Hematology Hospital from July 2017 to June 2018. ITP was diagnosed based on recently reported standards. Among the 50 patients, 25 were newly diagnosed  with a median platelet count of 19 × 109/l (range: 1–44 × 109/l) at diagnosis, and the other 25 were chronic ITP patients  with a median platelet count of 44 × 109/l at diagnosis (range: 3–165 × 109/l).Treatment response was determined based on a consensus of Chinese haematologists . That isThe control group comprised of healthy, age-matched subjects. The baseline characteristics of the patients and controls are summarized in 7/ml [The EDTA blood was centrifuged at 150 × g for 10 minutes to obtain platelet-rich plasma samples, which were then transferred to polypropylene test tubes. PRP was centrifuged at 2,300 × g for 5 minutes to obtain platelet pellets, which were then resuspended in ammonium oxalate (1%) for erythrocyte lysis. After centrifuging at 1100 × g for 5 minutes to discard the supernatant, the pellet was washed twice in phosphate-buffered saline (PBS) containing EDTA . The concentration of platelets in PBS-EDTA buffer was 1 × 107/ml –26.The platelets were stained with Anti-CD41-FITC , Anti-CD61-APC , and Anti-APRIL-PE (R&D Systems) at room temperature for 30 minutes. The platelets were then washed twice with PBS-EDTA buffer and resuspended in 0.5 ml of PBS-EDTA buffer and immediately analyzed on a flow cytometer. The platelets were sorted on a FACS Canto II instrument , and data were analyzed with the FlowJo v.10.0 software .Peripheral blood was collected into EDTA-anticoagulant vacuum tubes. PBMCs were isolated using lymphoprep density gradient centrifugation . PBMCs cells were stained with various combinations of monoclonal antibody (mAb) for 30 min on ice in staining buffer (1% BSA in PBS). The directly conjugated mAbs used were anti-CD19-FITC (BioLegend), anti-CD3-APC (BioLegend), anti-CD14-PECY7 (BioLegend) and anti-APRIL-PE (R&D Systems). Stained cells were washed twice with 2 ml PBS buffer, fixed in 1% paraformaldehyde, and analyzed within 24 h. The data were analyzed with the FlowJo v.10.0 software .Peripheral blood was collected into EDTA-anticoagulant vacuum tubes. Plasma was separated from blood cells by centrifugation  and was frozen at −80°C until analysis. PBMCs were isolated using lymphoprep density gradient centrifugation . APRIL levels in patient and control plasma were detected by antigen capture enzyme-linked immunosorbent assay using a commercial kit (BioLegend) according to the manufacturer's instructions. Plasma APRIL concentration was calculated from a standard curve generated using purified human APRIL supplied with the kit.t-test was used for comparison of sample mean within the group. The Pearson correlation coefficient or Spearman rank correlation was used to analyze the correlation between variables. The results before and after treatment were analyzed by paired t-test. P < 0.05 was considered statistically significant. All experimental analyses were performed using the GraphPad Prism 7.0 software.Data are expressed as mean ± standard error of the mean and were analyzed using SPSS v.13.5 . Factor variance analysis or The APRIL level of both CR and NR ITP patients is shown in Compared with isotype controls, surface APRIL could be clearly detected only in platelets in all ITP patients and normal controls. The mean fluorescence intensity (MFI) of platelet surface APRIL staining in ITP patients and normal controls was 631 and 1241, respectively. In contrast, little surface APRIL was observed in other immune cells. Specifically, the MFI of APRIL on the surface of CD19 + B cells in ITP patients and normal controls was 230 and 225, on that of CD3 + T cells being 224 and 272 and on that of CD14 + monocytes being 184 and 282, respectively. P < 0.001) and chronic (P < 0.001) ITP patients relative to controls.Compared with isotype controls, surface APRIL can be clearly detected on the surface of gated platelets in all ITP patients and normal controls . A graphr = 0.1811, P = 0.0013)  .n = 18). In patients with complete remission, APRIL MFI on platelet was higher after treatment than before treatment in the CR group   (The changes of platelet surface APRIL were monitored before and after treatment in active ITP patients ( 0.0204) , whereas 0.9964) .P < 0.001) and that in chronic ITP patients was 10.0 ± 0.96 ng/ml . These values were significantly higher than that of the normal control group.The average serum APRIL concentration in the normal control group was 6.87 ± 0.58 ng/ml (range: 5.67–8.07 ng/ml) . The mear = −6.123, P = 0.0007)    and the NR group   . Platele 0.0424) . After t 0.3464) . There w Figures .In this study, we investigated the relationship between APRIL and ITP, generating some interesting results. We began with exploring whether and how APRIL was expressed on the surface of immune cells and platelets in ITP patients and normal controls. We found that APRIL was saliently expressed only on the surface of platelets in both ITP patients and normal controls. Compared with controls, platelet surface APRIL was significantly reduced in ITP patients. This trend was obviously reversed in ITP patients with complete remission after treatment. In contrast, for patients who did not respond to treatment, the platelet surface APRIL level remained similar before and after treatment. We interpret our findings below.We were triggered by recent studies that detected surface APRIL in patients who suffered from diseases such as leukemia and RA –22 to asOur finding that the platelet surface APRIL level in the ITP patients fell significantly below the controls may be explained by five reasons. Firstly, it may be related to the imbalance of furin, since APRIL is processed intracellularly by furin convertase. While research on ITP on this issue is lacking, it has been reported that furin protease dysregulation is associated with increased levels of BAFF and APRIL in immune cells of patients with advanced atherosclerotic plaque . The secβ), TNF and its receptors (TNFR-1 and TNFR-2), and Glycoprotein V [An additional explanation for the plummet of platelet surface APRIL level in the ITP patients may be related to ADAM17 . It has rotein V –35. SincNext, we explored how treatment affected the expression of surface APRIL in ITP patients. We found that, for CR patients, the platelet surface APRIL level after treatment was significantly higher than that before treatment. In contrast, for NR patients, platelet surface APRIL level remained similar before and after treatment. This finding seems to suggest that surface APRIL may also play a role in immunoregulation together with soluble APRIL, again challenging Lopez-Fraga et al. who suggested that only soluble APRIL has an immunoregulation role . At presTo sum up, membrane-bound APRIL was only expressed on the surface of platelets in both ITP patients and normal controls with platelet surface APRIL in the former significantly lower than that in the latter. Platelet surface APRIL was reversed by treatment for CR patients but not for NR ones. Platelet surface APRIL may be a source of the excessive APRIL in the plasma of ITP patients. Aberrant APRIL expression on the surface of platelets is likely linked to autoimmune response in ITP patients. Our findings not only suggest important future research directions but also are clinically significant.