Developmental changes in human megakaryopoiesis.

BACKGROUND: The molecular bases of the cellular changes that occur during human megakaryocyte (MK) ontogeny remain unknown, and may be important for understanding the significance of MK differentiation from human embryonic stem cells (hESCs) METHODS: We optimized the differentiation of MKs from hESCs, and compared these with MKs obtained from primary human hematopoietic tissues at different stages of development. RESULTS: Transcriptome analyses revealed a close relationship between hESC-derived and fetal liver-derived MKs, and between neonate-derived and adult-derived MKs. Major changes in the expression profiles of cell cycle and transcription factors (TFs), including MYC and LIN28b, and MK-specific regulators indicated that MK maturation progresses during ontogeny towards an increase in MK ploidy and a platelet-forming function. Important genes, including CXCR4, were regulated by an on-off mechanism during development. DISCUSSION: Our analysis of the pattern of TF network and signaling pathways was consistent with a growing specialization of MKs towards hemostasis during ontogeny, and support the idea that MKs derived from hESCs reflect primitive hematopoiesis.

Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles.

Prostate cancer represents the first leading cause of cancer among western male population, with different clinical behavior ranging from indolent to metastatic disease. Although many molecules and deregulated pathways are known, the molecular mechanisms involved in the development of prostate cancer are not fully understood. The aim of this study was to explore the molecular variation underlying the prostate cancer, based on microarray analysis and bioinformatics approaches. Normal and prostate cancer tissues were collected by macrodissection from prostatectomy pieces. All prostate cancer specimens used in our study were Gleason score 7. Gene expression microarray (Agilent Technologies) was used for Whole Human Genome evaluation. The bioinformatics and functional analysis were based on Limma and Ingenuity software. The microarray analysis identified 1119 differentially expressed genes between prostate cancer and normal prostate, which were up- or down-regulated at least 2-fold. P-values were adjusted for multiple testing using Benjamini-Hochberg method with a false discovery rate of 0.01. These genes were analyzed with Ingenuity Pathway Analysis software and were established 23 genetic networks. Our microarray results provide new information regarding the molecular networks in prostate cancer stratified as Gleason 7. These data highlighted gene expression profiles for better understanding of prostate cancer progression.

Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma.

BACKGROUND: Combination of age at diagnosis, stage and MYCN amplification stratifies neuroblastoma into low-risk and high-risk. We aimed to establish whether a microRNA (miRNA) signature could be associated with prognosis in both groups. METHODS: Microarray expression profiling of human miRNAs and quantitative reverse-transcriptase PCR of selected miRNAs were performed on a preliminary cohort of 13 patients. Results were validated on an independent cohort of 214 patients. The relationship between miRNA expression and the overall or disease-free survival was analysed on the total cohort of 227 patients using the log-rank test and the multivariable Cox proportional hazard model. RESULTS: A total of 15 of 17 miRNAs that discriminated high-risk from low-risk neuroblastoma belonged to the imprinted human 14q32.31 miRNA cluster and two, miR-487b and miR-410, were significantly downregulated in the high-risk group. Multivariable analyses showed miR-487b expression as associated with overall survival and disease-free survival in the whole cohort, independently of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival of the non-MYCN-amplified favourable neuroblastoma: localised (stage 1, 2 and 3) and stage 4 of infant <18 months. CONCLUSION: Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favourable neuroblastoma.

Genome wide expression profile in human HTR-8/Svneo trophoblastic cells in response to overexpression of placental alkaline phosphatase gene.

During pregnancy, placental growth allows the adaptation of the feto-maternal unit to fetal requirements. Placental alkaline phosphatase (PLAP) is a phosphomonoesterase produced increasingly until term by the placenta and also ectopically in some tumors. To precise the role of this enzyme in the placenta, we analyzed the genome wide expression profile of HTR-8/Svneo trophoblastic cells after overexpression of the alkaline phosphatase gene (ALPP). We showed that ALPP overexpression mainly altered expression of genes implicated in cellular growth and proliferation. These results were confirmed by the study of cellular effects in HTR-8/Svneo cells overexpressing ALPP and in HTR-8/Svneo cells in which ALPP expression was suppressed by siRNA. We showed that PLAP exerts a positive effect on DNA replication and acts as a proliferative factor in trophoblastic cells.

Determination of metabolic fluxes during glucose catabolism in Propionibacterium freudenreichii subsp. shermanii.

In vivo 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the pathways of glucose metabolism, non-invasively, in living cell suspensions of Propionibacterium freudenreichii subsp. shermanii. This species is the main ripening flora of the Swiss-type cheeses and is widely used as propionic acid and vitamin B12 industrial producer. The flow of labelled [1-13C]glucose was monitored in living cell suspensions and enrichment was detected in main products like [1-13C]glycogen, [6-13C]lycogen, [1-13C]trehalose, [6-13C]trehalose, [1-13C]propionate, [2-13C]propionate, [3-13C]propionate, [1-13C]acetate, [2-13C]acetate, [1-13C]succinate, [2-13C]succinate and [1-13C]CO2. alpha and beta glucose consumption could be examined separately and were catabolized at the same rate. Three intermediates were also found out, namely [1-13C]glucose-6-phosphate, [6-13C]glucose-6-phosphate and [1-13C]glucose-1-phosphate. From the formation of intermediates such as [6-3C]glucose-6-phosphate and products like [6-13C]glycogen from [1-13C]glucose we concluded the bidirectionality of reactions in the first part of glycolysis and the isomerization at the triose-phosphate level. Comparison of spectra obtained after addition of [1-13C]glucose or [U-12C]glucose revealed production of [1-13C]CO2 which means that pentose phosphates pathway is active under our experimental conditions. From the isotopic pattern of trehalose, it could be postulated that trehalose biosynthesis occurred either by direct condensation of two glucose molecules or by gluconeogenesis. A chemically defined medium was elaborated for the study and trehalose was the main osmolyte found in the intracellular fraction of P. shermanii grown in this medium.