A new prognostic model of esophageal squamous cell carcinoma based on Cloud-least squares support vector machine.

Background: In view of the low accuracy of the prognosis model of esophageal squamous cell carcinoma (ESCC), this study aimed to optimize the least squares support vector machine (LSSVM) algorithm to determine the uncertain prognostic factors using a Cloud model, and consequently, to establish a new high-precision prognosis model of ESCC. Methods: We studied 4,771 ESCC patients(training samples) from the Surveillance, Epidemiology, and End Results (SEER) database and 635 ESCC patients(validation samples) from the Henan Provincial Center for Disease Control and Prevention (HCDC) database, with the same exclusion criteria and inclusion criteria for both databases, and obtained permission to obtain a research data file in the SEER database from the National Cancer Institute. The independent risk factors were analyzed using the log-rank method, survival curves, univariate and multivariate Cox analysis. Finally, the independent prognostic factors were used to construct the nomogram, random forest and Cloud-LSSVM prognostic models were utilized for validation. Results: The overall median survival time of the SEER database was 14 months (HCDC samples was 46 months), the mean survival time was 26.5 months (HCDC samples was 36.8 months), and the 3-year survival rate was 65.8%. This is because most of the patients with Henan samples are early ESCC, and most of the Seer patients are T3 and T4 people. The multivariate Cox analysis showed that age at diagnosis (P<0.001), sex (P=0.001), race (P=0.002), differentiation grade (P<0.001), pathologic T category (P<0.001), and pathologic M category (P<0.001) were the factors affecting the prognosis of ESCC patients. The SEER data and HCDC database results showed that the accuracy of the Cloud-LSSVM (C-index =0.71, 0.689) model is higher than the differentiation grade (C-index =0.548, 0.506), random forest (C-index =0.649, 0.498), and nomogram (C-index =0.659, 0.563). This new model can realize the unity of the randomness and fuzziness of the Cloud model and utilize the powerful learning and non-linear mapping abilities of LSSVM. Conclusions: Due to the difference of clans between training samples and test samples, the accuracy of prediction is generally not high, but the accuracy of Cloud-LSSVM model is much higher than other models. The new model provides a clear prognostic superiority over the random forest, nomogram, and other models.

PEI-Fe3O4 Nanoparticles for Human Amniotic Epithelial Cells Labeling

Objective To label human amniotic epithelial cells(hAECs) by using PEI-Fe3O4 nanoparticles. Methods The PEI-Fe3O4 nanoparticles were characterized by using transmission electron microscopy and dynamic light scattering. The primary cultured hAECs were labeled with the nanoparticles,and the labeling efficiency was evaluated by Prussian blue staining. The cell survival rate and viability were tested by using placenta blue staining and CCK-8 assay,respectively. Results The PEI-Fe3O4 nanoparticles were compact spheres with an average particle size of 13 nm,a hydrodynamic radius of 17.56 nm,and a zeta potential of+34.5 mV. The labeling efficiency of the nanoparticles on hAECs reached 91% when the concentrations were greater than 20 µg/ml. When the concentrations of nanoparticles were at 50 µg/ml(t=16.37,P<0.0001;t=10.39,P<0.0001) and 100 µg/ml(t=29.89,P<0.0001;t=16.86,P<0.0001),the cell survival rates and cell viabilities were significantly reduced versus controls. Conclusion The PEI-Fe3O4 nanoparticles can be used for labeling hAECs without obvious cytotoxicity at its working concentration.

Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells.

Identifying molecular mechanisms that regulate insulin expression in bone marrow-derived mesenchymal stem cells (bmMSCs) can provide clues on how to stimulate the differentiation of bmMSCs into insulin-producing cells (IPCs), which can be used as a therapeutic approach against type 1 diabetes (T1D). As repression factors may inhibit differentiation, the efficiency of this process is insufficient for cell transplantation. In this study, we used the mouse insulin 2 (Ins2) promoter sequence and performed a DNA affinity precipitation assay combined with liquid chromatography-mass spectrometry to identify the transcription factor, chicken ovalbumin upstream promoter transcriptional factor I (COUP-TFI). Functionally, bmMSCs were reprogrammed into IPCs via COUP-TFI suppression and MafA overexpression. The differentiated cells expressed higher levels of genes specific for islet endocrine cells, and they released C-peptide and insulin in response to glucose stimulation. Transplantation of IPCs into streptozotocin-induced diabetic mice caused a reduction in hyperglycemia. Mechanistically, COUP-TFI bound to the DR1 (direct repeats with 1 spacer) element in the Ins2 promoter, thereby negatively regulating promoter activity. Taken together, the data provide a novel mechanism by which COUP-TFI acts as a negative regulator in the Ins2 promoter. The differentiation of bmMSCs into IPCs could be improved by knockdown of COUP-TFI, which may provide a novel stem cell-based therapy for T1D.

MiR-378b Promotes Differentiation of Keratinocytes through NKX3.1.

MicroRNA (miRNA) is a kind of short non-coding RNA, involved in various cellular processes. During keratinocyte differentiation, miRNAs act as important regulators. In this study, we demonstrated by microarray assay that the expression of miR-378b significantly increased during keratinocytes differentiation. Our findings showed that miR-378b could inhibit proliferation, migration and differentiation in keratinocytes. Luciferase reporter assays showed that miR-378b directly target NKX3.1. Silencing of NKX3.1 could coincide with the effects of miR-24 overexpression. In conclusion, our results demonstrate miR-378b promote keratinocytes differentiation by targeting NKX3.1. Manipulation of miR-378b may afford a new strategy to clinic treatment of skin injury and repair.

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii.

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.

[Expression and clinical significance of beta-catenin in multiple myeloma].

BACKGROUND & OBJECTIVE: beta-catenin is the pivotal regulator in Wnt pathway and in charge of cellular adhesion and signal conduction. Overexpression of beta-catenin has been observed in various human tumors. This study was to investigate the expression and clinical significance of beta-catenin in multiple myeloma (MM). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect mRNA and protein expression of beta-catenin in bone marrow samples from 12 newly diagnosed MM patients, 14 relapsed/refractory MM patients, and 11 healthy donors. The clinical data and treatment outcomes of the MM patient were also analyzed. RESULTS: The positive rate and the expression level of beta-catenin mRNA were significantly lower in healthy donors than in newly diagnosed patients and relapsed/refractory patients (27.3% vs. 75.0% and 100%, P=5.01e(-4); 0.35+/-0.17 vs. 0.72+/-0.11 and 0.85+/-0.16, P=5.88e(-5)); the mRNA level of beta-catenin was significantly lower in newly diagnosed patients than in relapsed/refractory patients (P=0.045). beta-catenin protein was not detected in healthy donors; while the positive rate and the expression level of beta-catenin protein were significantly lower in newly diagnosed patients than in relapsed/refractory patients (50.0% vs. 85.7%, 0.32+/-0.11 vs. 0.21+/-0.08, P=0.039). In the 10 newly diagnosed MM patients with evaluable treatment outcomes, the positive rate of beta-catenin protein was significant lower in the 7 patients without response than in the 3 patients showed response (14.3% vs. 100%, P=0.033). The positive rate of beta-catenin protein was significant higher in the 16 Durie/Salmon stage III patients than in the 10 stage II patients (87.5% vs. 40.0%, P=0.026), and significant higher in ISS stage III patients than in stage I and II patients (100% vs. 45.5% and 33.3%, P=0.006, P=0.032). The protein level of beta-catenin was positively correlated to serum levels of beta2-MG (r=0.688, P=0.002) and lactate dehydrogenase (LDH) (r=0.502, P=0.034). CONCLUSION: The expression of beta-catenin is related with treatment outcome, Durin-Salmon stage and ISS stage of MM, and is positively correlated to serum levels of LDH and beta2-MG.

[Effect of silencing survivin gene on sensitivity of myeloma cells to adriamycin].

BACKGROUND & OBJECTIVE: Survivin, a member of the inhibitors of apoptosis protein family (IAPs), is overexpressed in many cancers, but not in normally differentiated adult tissues. It is known to be involved in poor prognosis and resistance to chemotherapy and radiotherapy in many cancers. This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human myeloma cell line KM3, observe its effects on apoptosis of KM3 cells and sensitivity of KM3 cells to adriamycin (ADM). METHODS: Small interfering RNA (siRNA) targeting survivin mRNA was designed and in vitro chemosynthesized, and transfected into KM3 cells. Survivin mRNA and protein levels in KM3 cells were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot 24, 48, and 72 h after transfection. The apoptosis of KM3 cells was observed under fluorescent microscope before and after transfection. The sensitivity of KM3 cells to ADM before and after transfection was assessed. RESULTS: The mRNA level of survivin was down-regulated by (47.0+/-4.3)%, (81.4+/-6.2)%, and (49.1+/-7.9)% of control at 24, 48, and 72 h after siRNA transfection, respectively; while the protein level of survivin was down-regulated by (39.6+/-3.8)%, (56.4+/-6.7)%, and (68.2+/-5.1)% of control, respectively. Under fluorescent microscope, the apoptosis rate of KM3 cells was (28+/-7)% at 48 h after transfection of survivin siRNA, which was significantly higher than that of control cells after transfection of negative siRNA (P<0.05). The 50% inhibition concentration (IC(50)) of ADM to KM3 cells was decreased from (1.12+/-0.14) micromol/L to (0.31+/-0.03) micromol/L. CONCLUSION: Down-regulation of survivin expression in KM3 cells by siRNA could effectively induce apoptosis of KM3 cells and increase their sensitivity to ADM.

[Expression and clinical significance of survivin in bone marrow cells of multiple myeloma patients].

BACKGROUND & OBJECTIVE: Survivin, a member of the inhibitors of apoptosis protein family (IAPs), is overexpressed in many cancers, but not in normal differentiated adult tissues. It is related to poor prognosis and resistance to chemotherapy and radiotherapy in many cancers. This study was designed to detect the expression of Survivin in bone marrow of healthy people, newly diagnosed and relapsed/refractory multiple myeloma (MM) patients, and MM cell line KM3, to explore its correlation to drug resistance and prognosis of MM patients. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect mRNA and protein expression of Survivin in bone marrow samples of 17 newly diagnosed MM patients, 12 relapsed /refractory MM patients, and 9 healthy people, and myeloma cell line KM3. RESULTS: KM3 cells expressed Survivin strongly. The positive rates and the expression levels of Survivin mRNA were significantly lower in healthy people than in newly diagnosed patients and relapsed/refractory patients (22.2 % vs. 70.5 % and 83.3 %, 0.06+/-0.04 vs. 0.31+/-0.14 and 0.69+/-0.24, P<0.01), and were significantly lower in newly diagnosed patients than in relapsed/refractory patients (P<0.05). Survivin protein were not detected in all of the healthy people; while the positive rates of Survivin were 41.4% in newly diagnosed patients and 58.3% in relapsed/refractory patients (P>0.05), and its expression levels was significantly higher in relapsed/refractory patients than in newly diagnosed patients (0.21+/-0.12 vs. 0.11+/-0.07, P<0.05). Among the 7 Survivin-positive newly diagnosed patients, 4 achieved partial remission (PR), 1 achieved MR, and 2 showed no change (NC); among the 10 Survivin-negative newly diagnosed patients, 2 achieved complete remission (CR), 4 achieved PR, 2 achieved MR, and 2 showed NC. The effective (CR+PR+MR) rate was slightly higher in Survivin-negative patients than in Survivin-positive patients (80.0% vs. 71.4%). CONCLUSION: High expression of Survivin may be relates to drug resistance in some patients. Since Survivin only expressed in bone marrow cells of multiple myeloma, but not in healthy people, it can be a new target to reverse drug resistance in myeloma cells.