Les oncogènes NUP98-PHF23 et NUP98-HOXD13 confèrent un potentiel aberrant d’auto-renouvellement aux progéniteurs thymiques.

L’initiation de la leucémogénèse dans la leucémie aigue lymphoblastique (LAL)-T résulte de l’activation aberrante de facteurs de transcription de la lignée lymphocytaire T. Nous démontrons que les gènes de fusion NUP98-PHF23 (NP23) et NUP98-HOXD13 (NHD13) reprogramment les thymocytes normaux en cellules souches pré-leucémiques (CS-préL) possédant un potentiel aberrant d’auto-renouvellement. Basé sur des essais de clonalité performés sur des thymocytes transplantés en série, nous avons découvert que cette population est hiérarchisée similairement aux cellules souches hématopoïétiques normales. Ces CS-préL dévoilent un enrichissement du compartiment de précurseurs thymiques immatures KIT+ où les deux oncogènes, NP23 et NHD13, activent des gènes impliqués dans l’autorenouvellement, incluant Hoxa9, Hoxa10, Lyl1 et Hhex. De plus, l’activité d’autorenouvellement est abrogée par les ARN interférents contre Lyl1 et Hhex, indiquant leur implication fonctionnelle en aval de NP23 et NHD13. Puisque ces gènes sont aussi activés en aval de trois autres oncogènes dans la LAL-T, SCL/TAL1, LMO1 et LMO2, nous concluons que les niveaux d’activation de Lyl1 et Hhex fixent le seuil de reprogrammation des thymocytes normaux en CS-préL. Malgré l'efficacité des traitements de chimiothérapie actuels à diminuer la masse tumorale, les CS-préL sont épargnées, pouvant mener à des rechutes. Nos résultats répondent à ce besoin et proposent de nouvelles avenues permettant de cibler les CS-préL du compartiment de thymocytes immatures dans la LAL-T.

Reverse pivoting in conceptual information systems

In database marketing, the behavior of customers is analyzed by studying the transactions they have performed. In order to get a global picture of the behavior of a customer, his single transactions have to be composed together. In On-Line Analytical Processing, this operation is known as reverse pivoting. With the ongoing data analysis process, reverse pivoting has to be repeated several times, usually requiring an implementation in SQL. In this paper, we present a construction for conceptual scales for reverse pivoting in Conceptual Information Systems, and also discuss the visualization. The construction allows the reuse of previously created queries without reprogramming and offers a visualization of the results by line diagrams.

AID enzymatic activity is inversely proportional to the size of cytosine C5 orbital cloud

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID’s enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H . F . methyl .. hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.

Adult palatum as a novel source of neural crest-related stem cells

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies.

Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.

Production of Bovine Hand-Made Cloned Embryos by Zygote-Oocyte Cytoplasmic Hemi-complementation

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; handmade cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus)

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such `labour-intensive technology`.

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Resposta de Chromobacterium Violaceum cultivada em alta concentração de ferro

The Chromobacterium violaceum is a β-proteobacterium Gram-negative widely found in tropical and subtropical regions, whose genome was sequenced in 2003 showing great metabolic versatility and biotechnological and pharmaceutical potential. Given the large number of ORFs related to iron metabolism described in the genome of C. violaceum, the importance of this metal for various biological processes and due to lack of data about the consequences of excess of iron in free-living organisms, it is important to study the response mechanism of this bacterium in a culture filled with iron. Previous work showed that C. violaceum is resistant to high concentrations of this metal, but has not yet been described the mechanism which is used to this survival. Thus, to elucidate the response of C. violaceum cultured in high concentrations of iron and expecting to obtain candidate genes for use in bioremediation processes, this study used a shotgun proteomics approach and systems biology to assess the response of C. violaceum grown in the presence and absence of 9 mM of iron. The analysis identified 531 proteins, being 71 exclusively expressed by the bacteria grown in the presence of the metal and 100 just in the control condition. The increase in expression of proteins related to the TCA cycle possibly represents a metabolic reprogramming of the bacteria caused by high concentration of iron in the medium. Moreover, we observed an increase in the activity assay of superoxide dismutase and catalase as well as in Total Antioxidant Activity assay, suggesting that the metal is inducing oxidative stress in C. violaceum that increases the levels of violacein and antioxidant enzymes to better adapt to the emerging conditions. Are also part of the adaptive response changes in expression of proteins related to transport, including iron, as well as an increased expression of proteins related to chemotaxis response, which would lead the bacteria to change the direction of its movement away from the metal. Systems Biology results, also suggest a metabolic reprogramming with mechanisms coordinated by bottleneck proteins involved in transcription (GreA), energy metabolism (Rpe and TpiA) and methylation (AhcY)

Xenooplasmic Transfer between Buffalo and Bovine Enables Development of Homoplasmic Offspring

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemically Assisted Enucleation Results in Higher G6PD Expression in Early Bovine Female Embryos Obtained by Somatic Cell Nuclear Transfer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Mechanism of Oocyte Activation Influences the Cell Cycle-Related Genes Expression During Bovine Preimplantation Development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morpho-histochemical characterization of salivary gland cells of males of the tick Rhipicephalus sanguineus (Acari: Ixodidae) at different feeding stages: description of new cell types

This study describes the changes undergone by cells of the salivary glands of unfed and feeding (at day two and four post-attachment) Rhipicephalus sanguineus males, as well as new cell types. In unfed males, types I and II acini are observed with cells undifferentiated, undefined 1 and 2 (the latter, with atypical granules), a, c1 and c3; type III is composed of cells d and e; and type IV present cells g. In males at day two post-attachment, type I acini exhibit the same morphology of unfed individuals. An increase in size is observed in types II, III, and IV, as cells are filled with secretion granules. Some granules are still undergoing maturation. In type II acinus, cells a, b and c1-c8 are observed. Cells c7 and c8 are described for the first time. Cells c7 are termed as such due to the addition of polysaccharides in the composition of the secretion granules (in unfed individuals, they are termed undefined 1). Type III acini exhibit cells d and e completely filled with granules, and in type IV, cells g contain granules in several stages of maturation. In males at day four post-attachment, type I acini do not exhibit changes. Granular acini exhibit cells with fewer secretion granules, which are already mature. In type II acini, cells a, b, c1-c5 are present, type III exhibit cells d and e, and type IV contain cells g with little or no secretion. This study shows that in the salivary glands of R. sanguineus males, cells a, c1, and c3 of type II acinus, and cells d and e of type III do not exhibit changes in granular content, remaining continuously active during the entire feeding period. This indicates that during the intervals among feeding stages, gland cells reacquire the same characteristics found in unfed individuals, suggesting that they undergo reprogramming to be active in the next cycle.