Inferring cancer common and specific gene networks via multi-layer joint graphical model.

Cancer is a complex disease caused primarily by genetic variants. Reconstructing gene networks within tumors is essential for understanding the functional regulatory mechanisms of carcinogenesis. Advances in high-throughput sequencing technologies have provided tremendous opportunities for inferring gene networks via computational approaches. However, due to the heterogeneity of the same cancer type and the similarities between different cancer types, it remains a challenge to systematically investigate the commonalities and specificities between gene networks of different cancer types, which is a crucial step towards precision cancer diagnosis and treatment. In this study, we propose a new sparse regularized multi-layer decomposition graphical model to jointly estimate the gene networks of multiple cancer types. Our model can handle various types of gene expression data and decomposes each cancer-type-specific network into three components, i.e., globally shared, partially shared and cancer-type-unique components. By identifying the globally and partially shared gene network components, our model can explore the heterogeneous similarities between different cancer types, and our identified cancer-type-unique components can help to reveal the regulatory mechanisms unique to each cancer type. Extensive experiments on synthetic data illustrate the effectiveness of our model in joint estimation of multiple gene networks. We also apply our model to two real data sets to infer the gene networks of multiple cancer subtypes or cell lines. By analyzing our estimated globally shared, partially shared, and cancer-type-unique components, we identified a number of important genes associated with common and specific regulatory mechanisms across different cancer types.

Pancreatic transcription factors containing protein transduction domains drive mouse embryonic stem cells towards endocrine pancreas.

Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types, including stem cells. Here we generated pancreatic transcription factors containing PTD sequences and administered these to endoderm enriched mouse embryonic stem (ES) cells under conditions that were designed to mimic the pattern of expression of these factors in the developing pancreas. The ES cells were first cultured as embryoid bodies and treated with Activin A and Bone morphogenetic protein 4 (BMP4) to promote formation of definitive endoderm. Cells were subsequently plated as a monolayer and treated with different combinations of the modified recombinant transcription factors Pdx1 and MafA. The results demonstrate that each transcription factor was efficiently taken up by the cells, where they were localized in the nuclei. RT-qPCR was used to measure the expression levels of pancreatic markers. After the addition of Pdx1 alone for a period of five days, followed by the combination of Pdx1 and TAT-MafA in a second phase, up-regulation of insulin 1, insulin 2, Pdx1, Glut2, Pax4 and Nkx6.1 was observed. As assessed by immunocytochemistry, double positive insulin and Pdx1 cells were detected in the differentiated cultures. Although the pattern of pancreatic markers expression in these cultures was comparable to that of a mouse transformed ß-cell line (MIN-6) and human islets, the expression levels of insulin observed in the differentiated ES cell cultures were several orders of magnitude lower. This suggests that, although PTD-TFs may prove useful in studying the role of exogenous TFs in the differentiation of ES cells towards islets and other pancreatic lineages, the amount of insulin generated is well below that required for therapeutically useful cells.

Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors.

The AR42J-B13 rat pancreatic acinar cell line was used to identify pancreatic transcription factors and exogenous growth factors (GFs) that might facilitate the reprogramming of exocrine cells into islets. Adenoviruses were used to induce exogenous expression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4. Individually Pdx1, MafA and Pax4 had no effect on the expression of endocrine markers, whilst adeno-Ngn3 on its own increased the expression of Pax4, Ngn3 and NeuroD. In combination the four TFs had a significant effect on the expression of insulin 1 and 2 that was associated with a change in cell morphology from a rounded to a spindle-like shape. Amongst a range of growth factors, Betacellulin and Nicotinamide were shown to enhance the effects of the four TFs. The presence of adeno-Pax4 in the differentiation cocktail was important in limiting the expression of glucagon and in generating glucose sensitive insulin secretion. Further experiments asked whether the adenoviral TFs could be replaced by protein transduction domain (PTD)-containing TFs. The results showed that the PTD-TFs could mimic in part the effects of the adeno-TFs, but the resultant cells did not undergo the important morphological change associated with differentiation to endocrine lineages and levels of endogenous markers were very much lower. In summary, the results describe a cocktail of four TFs and two GFs that can be used to induce formation of glucose sensitive insulin secreting cells from ARJ42 cells, and demonstrate that it would be difficult to replace adenoviral transduction with PTD-TFS.

Murine embryonic stem cell-derived hepatocytes correct metabolic liver disease after serial liver repopulation.

Although embryonic stem (ES) cell-derived hepatocytes have the capacity for liver engraftment and repopulation, their in vivo hepatic function has not been analyzed yet. We aimed to determine the metabolic function and therapeutic action of ES cell-derived hepatocytes after serial liver repopulations in fumaryl acetoacetate hydrolase knockout (Fah(-/-)) mice. Albumin expressing (Alb(+)) cells were obtained by hepatic differentiation of ES cells using two frequently reported methods. After transplantation, variable levels of liver repopulation were found in Fah(-/-) mice recipients. FAH expressing (FAH(+)) hepatocytes were found either as single cells or as nodules with multiple hepatocytes. After serial transplantation, the proportion of the liver that was repopulated by the re-transplanted FAH(+) hepatocytes increased significantly. ES cell-derived FAH(+) hepatocytes were found in homogenous nodules and corrected the liver metabolic disorder of Fah(-/-) recipients and rescued them from death. ES cell-derived hepatocytes had normal karyotype, hepatocytic morphology and metabolic function both in vitro and in vivo. In conclusion, ES cell-derived hepatocytes were capable of liver repopulation and correction of metabolic defects after serial transplantation. Our results are an important piece of evidence to support future clinical applications of ES cell-derived hepatocytes in treating liver diseases.

Phylogenetic analysis of Sarcocystis nesbitti (Coccidia: Sarcocystidae) suggests a snake as its probable definitive host.

Sarcocystis nesbitti was first described by Mandour in 1969 from rhesus monkey muscle. Its definitive host remains unknown. 18S rRNA gene of S. nesbitti was amplified, sequenced, and subjected to phylogenetic analysis. Among those congeners available for comparison, it shares closest affinity with those species of Sarcocystis which use snakes as definitive hosts. We therefore hypothesize that a snake may serve as the definitive host for S. nesbitti.

Expression and cytotoxicity of EGFP-labeled D-amino acid oxidase in HeLa cells.

To observe the expression of R. gracilis D-amino acid oxidase (DAAO) gene labeled by EGFP in HeLa cells and how it mediates cytotoxicity, DAAO cDNA and EGFP gene were cloned into Eukaryotic expression vector pIRES to construct DAAO gene expression vector pIRES-DAAO. HeLa cells were transfected with pIRES-DAAO in vitro. Expression of EGFP gene in HeLa-D cells was observed under a fluorescent microscope. The efficiency of transfection was analyzed by flow cytometry (FCM). The fluorescent cells were screened by FCM and were named HeLa-D. The activity of HeLa-D cells was detected by MTT colorimetric assay after they were treated with D-Ala at various concentrations. The results indicted that expression of EGFP gene in HeLa-D cells was seen under fluorescent microscope and HeLa-D cells were screened by FCM. Apparently,the prodrug D-Ala killed HeLa-D cells. These results demonstrate that EGFP gene can be regarded as a reporter gene to screen the cells transduced with DAAO quickly, and DAAO/D-Ala suicide gene system may prove helpful in gene therapy of cancer.