Commitment of hematopoietic stem cells in avian and mammalian embryos: an ongoing story

During ontogeny, hematopoietic stem cells (HSC) become committed outside of hematopoietic organs. Once held to emerge from the yolk sac, they are currently thought to originate from the early aorta. However we now show that the allantois in the avian embryo and the placenta in the mouse embryo produce HSC in very large numbers. These sites thus appear to have a central role in the process of HSC emergence.     

Molecular characterization of gilthead sea bream (Sparus aurata) lipoprotein lipase. Transcriptional regulation by season and nutritional condition in skeletal muscle and fat storage tissues

Lipoprotein lipase (LPL) of gilthead sea bream (Sparus aurata) was cloned and sequenced using a RT-PCR approach completed by 3' and 5'RACE assays. The nucleotide sequence covered 1669 bp with an open reading frame of 525 amino acids, including a putative signal peptide of 23 amino acids long. Sequence alignment and phylogenetic analysis revealed a high degree of conservation among most fish and higher vertebrates, retaining the consensus sequence the polypeptide "lid", the catalytic triad and eight cysteine residues at the N-terminal region. A tissue-specific regulation of LPL was also found on the basis of changes in season and nutritional condition as a result of different dietary protein sources. First, the expression of LPL in mesenteric adipose tissue was several times higher than in liver and skeletal muscle. Secondly, the spring up-regulation of LPL expression in the mesenteric adipose tissue was coincident with a pronounced increase of whole body fat content. Thirdly, the highest expression of LPL in the skeletal muscle was found in summer, which may serve to cover the increased energy demands for muscle growth and protein accretion. Further, in fish fed plant-protein-based diets, hepatic LPL expression was up-regulated whereas an opposite trend was found in the mesenteric adipose tissue, which may contribute to drive dietary lipids towards liver fat storage. Finally, it is of interest that changes in circulating triglyceride (TG) levels support the key role of LPL in the clearance of TG-rich lipoproteins. This study is the first report in fish of a co-regulated expression of LPL in oxidative and fat storage tissues under different physiological conditions.     

Using genomic inbreeding coefficient estimates for homozygosity mapping of rare recessive traits: application to Taybi-Linder syndrome

The use of inbred patients whose exact genealogy may not be available is of primary interest in mapping genes involved in rare recessive diseases. We show here that this can be achieved by estimating inbreeding coefficients from the patients' genomic information and using these estimates to perform homozygosity mapping. We show the interest of the approach by mapping a gene for Taybi-Linder syndrome to chromosome 2q, with the use of a key patient with no genealogical information.     

Development of a multiple internal control for clinical diagnostic real-time amplification assays

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.