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Background: It is essential to discriminate low-grade (G1), intermediate-grade (G2) and high-grade (G3) rectal neuroendocrine tumors (R-NETs) due to the treatment decisions and postoperative management are totally different. CT histogram analysis, which is a novel method to provide objectively quantified assessment of tumor, may be helpful for differentiating the grade of R-NETs. Methods: A total of 61 (35 G1, 12 G2 and 14 G3) patients who underwent preoperative contrast-enhanced CT and treated with surgery to be confirmed as R-NETs were included in this study from January 2014 and May 2019. We accurately depicted the region of interest (ROI) and measured the CT texture parameters (mean, median, 10th, 25th, 75th, 90th percentiles, skewness, kurtosis and entropy) from arterial phase image by two radiologists. We calculated and compared these parameters between low-grade (G1), intermediate-grade (G2) and high-grade (G3) tumors by applying appropriate statistical method. We obtained the optimal parameters to identify G1 and G2/G3 by using receiver operating characteristic (ROC) curves. Findings: Histogram analysis of CT arterial phase imaging demonstrated significant differences between G1 and G2 or G3 group in several histogram parameters (mean, median, 10th, 25th, 75th and 90th percentiles) (all P 0.05). Skewness, kurtosis and entropy showed no significant differences among three groups ( P =0.449, 0.285, 0.057, respectively). ROC analysis showed a good predictive performance between G1 and G2/G3 and the mean generated the highest area under the curve (AUC = 0.832), followed by the median (AUC = 0.827). Interpretation: CT histogram parameters, especially mean and median, can be used as excellent indicators for predicting the grade (G1 and G2/G3) of rectal neuroendocrine tumors. Funding Statement: This study has received funding by the National Natural Science Foundation of China (Grant No.81771801,81801695, 81701657 and 81873623). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The Institutional Review Board of our hospital (Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology) approved this retrospective study and the requirement for patient informed consent was waived.

Background Small cell lung cancer (SCLC) is a malignant lung neuroendocrine tumor with early metastasis, rapid progression, and poor outcomes. Insulinoma-associated protein 1 (INSM1) has been an excellent marker for neuroendocrine (NE) differentiation and widely used in the diagnosis of NE neoplasms, including SCLC. However, its role beyond NE diagnostic marker remained little reported. Methods We examined immunohistochemical expression of INSM1 in 73 surgically resected SCLC, analyzed its prognostic value by Kaplan-Meier method, and investigated clinical-pathological features of INSM1 high SCLC. In vitro, We assessed INSM1 function on glucose intake, tumor migration, and Cisplatin resistance by 2-NBDG glucose uptake fluorescent assay, transwell assay, and ANNEXIN V/PI assay, respectively. In vivo, we evaluated the therapeutic value of metformin on reversing INSM1 induced chemoresistance by BALB/c nude mice xenograft tumor model. Results High INSM1 expression was correlated with lymph node metastasis (LNM) (p = 0.0005), later TNM stages (p = 0.0003), and predicted poor survival (Log-rank p = 0.038). Multivariate Cox analysis confirmed INSM1 as an independent prognostic factor in SCLC (p = 0.012, HR:3.195, 95%CI:1.288–7.927). Interestingly, LNM was correlated with worse prognosis only in patients received chemotherapy (Log-rank p = 0.027) rather than the others (Log-rank p = 0.40). In patients having LNM and treated with chemotherapy, high INSM1 was correlated with worse clinic outcome (Log-rank p = 0.009). In vitro, overexpression of INSM1 decreased AMPK-α expression as well as glucose intake, promoted tumor cell migration, and limited the apoptosis induced by Cisplatin, which all could be reversed by Metformin. In vivo, INSM1 overexpression also contributed to tumor growth beyond inducing Cisplatin resistance. Conclusion Our finding suggested INSM1 played more role than a NE marker, partly through down-regulating AMPK signal. INSM1 may serve as a novel prognostic marker and therapeutic target in SCLC.

Aims Previous studies have shown that stem cell factor (SCF) induces the migration of cardiac stem cells (CSCs) and helps to repair myocardial infarctions. Earlier studies on the migration mechanism only focused on the activation of kinases; here, we aimed to explore the functional role of protein phosphatase 2A (PP2A) in SCF-induced CSC migration. Main methods CSCs were treated with SCF, PP2A enzymatic activity was measured, the phosphorylation levels of PP2A, p38 MAPK and cofilin were evaluated using western blot. Transwell assay was used to determine the migratory ability of CSCs. Key findings In vitro, SCF induced the phosphorylation of p38 MAPK and cofilin, leading to the migration of CSCs. Cofilin acted as a downstream signal of p38 MAPK. PP2A was involved in this process. Further studies revealed that PP2A was inactivated via phosphorylation at Tyr307 by SCF and the inactivation/phosphorylation was mediated by activated p38 MAPK, as p38 MAPK inhibitor SB203580 or siRNA prevented SCF-induced inactivation and phosphorylation of PP2A. When CSCs were pretreated with PP2A inhibitor (okadaic acid, OA), SCF-induced CSC migration and the downstream signals were enhanced, and the enhancement was reversed when p38 MAPK was blocked. Additionally, co-immunoprecipitation showed a direct interaction of PP2A with p38 MAPK. Significance Our results indicated that PP2A regulated the SCF-induced activation of p38 MAPK/cofilin signaling pathway and subsequent migration of CSCs by interaction with p38 MAPK.

Background: The optimal sampling techniques for endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) remain unclear and have not been standardized. The aim of the study was to compare modified wet suction technique (MWST) and dry suction technique (DRST) for sampling the solid lesions in the pancreas, mediastinum, and abdomen. Methods: We performed a randomised controlled trial at four tertiary care centers in China. Consecutive patients with solid lesions referred for EUS-FNA were randomised in a ratio of 1:1 to receive either DRST or MWST as the first pass. For Group A, the pass sequence was DRST - MWST - DRST - MWST versus MWST - DRST - MWST - DRST for Group B. The outcome measures were the diagnostic yield and specimen quality. Findings: Between May 2016 and January 2018, 364 patients were screened for eligibility. 296 patients were recruited, 269 of whom (134 in Group A versus 135 in Group B) completed the trial. We demonstrated that the MWST group had a significantly better overall histological diagnostic accuracy (84·85%) than DRST group (73·19%, P =0·0013) for all solid lesions (n=269). In subgroup analysis, the MWST group reached a 91·59% histological diagnostic accuracy for non-pancreatic lesions (n=108). Furthermore, the MWST provided better specimen adequacy and less blood contamination than DRST. Interpretation: In this prospective study of sampling solid masses, we found that using MWST resulted in significantly better histological diagnostic accuracy and specimen quality than DRST. For many institutions where rapid on-site evaluation is not routinely available, the MWST within 2 passes of FNA reached a definitive histological diagnosis with no requirement of fine needle biopsy. Trial Registration: The study is registered on ClinicalTrials.gov (NCT02789371) and is completed. Funding Statement: National Natural Science Foundation of China. Declaration of Interests: All authors have declared that no competing interests exist. Ethics Approval Statement: The study was approved by the institutional review boards of all participating centers. Patients provided informed consent for participation in the study.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia.

Human mesenchymal stem cells (HMSCs) have been applied in various clinic settings. Ion channels play an important role in cellular physiology. However, the potential role of cationic channels in regulating the proliferation and migration properties of hMSCs remains to be determined. In the present study, the functional expression of ion channels in hMSCs was investigated by patch clamp. MTT assay and BrdU stainings were used to assess the proliferation of hMSCs. hMSC migration was evaluated by Transwell migration assays. The results show that sodium-, L-type calcium, potassium currents have been identified in hMSCs. TEA (K(+) channel blocker), nifedipine (Ca(2+) channel blocker) can inhibit both proliferation and migration of hMSCs. The increase of extracellular Ca(2+) concentration promoted both proliferation and migration of hMSCs. TTX, a Na(+) channel blocker, promoted cell proliferation but inhibited cell migration. Our data suggest that cationic channels (sodium, L-type calcium, potassium channels) play important roles in regulating proliferation and migration of hMSCs.

Background Hyperhomocysteinemia (HHcy) has been reported as an independent risk factor for coronary artery disease; however it is not clear regarding the action of HHcy on the homing of cardiac stem cells (CSCs) to the damaged myocardium and the consequent CSCs-mediated cardiac repair post myocardial infarction. Methods Sprague–Dawley (SD) rats were divided into 4 groups. HHcy was induced in the rats by a 6-week high-methionine diet. Rat heart MI model was developed by left coronary artery ligation. Immunofluorescence was used to examine the CSCs migration in vivo via injecting BrdU-labeled CSCs into AV-groove followed by a coronary ligation. Immunohistochemistry, western blot and ELISA analysis were carried out to detect the expression of stem cell factor (SCF) protein, and RT-PCR was conducted for the expression of SCF mRNA. Results On day 5 of MI model creation, accumulation of CSCs was significantly increased in the peri-infarcted area by the non-hyperhomocysteinemic rats, which led to an improvement of cardiac function at 3 weeks after MI. however, the accumulation of CSCs was markedly decreased by the hyperhomocysteinemic rats followed with the decline of cardiac function. SCF expression was also significantly decreased in the peri-infarcted area by the hyperhomocysteinemic rats compared to the non-hyperhomocysteinemic rats. The experiments in vitro confirmed that homocysteine (Hcy) decreased SCF expression via inhibition of TNF-α-induced activity of NF-κB, further reduced the migration of CSCs. Conclusion It demonstrated that hyperhomocysteinemia may significantly contribute to restrain CSCs-mediated cardiac repair by reducing SCF-induced homing of CSCs.

The TCF4/beta-catenin complex, the executor of canonical Wnt/beta-catenin signaling, is regulated by a variety of factors. Among these, Dishevelled (Dvl) is a critical regulator that releases beta-catenin from degradation and stabilizes TCF4/beta-catenin complex. Here, we report that DDIP (Dishevelled-DEP domain Interacting Protein, also named as Spats1, spermatogenesis associated, serine-rich 1), a novel protein that interacts with Dvl, regulates Wnt signaling. We provide evidence that DDIP suppresses Lef-1 luciferase reporter activity stimulated by Wnt1, Dvl2 or beta-catenin, interacts with the TCF4/beta-catenin complex, and disrupts the interaction of TCF4 and beta-catenin by promoting TCF4 degradation through the proteasome pathway. Our results indicate that DDIP is a negative regulator of the canonical Wnt signaling.

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.