High frequency of in situ hybridization on thin plastic sections of human placenta with a cDNA probe for beta hCG

We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.     

Novel ABCA3 mutations as a cause of respiratory distress in a term newborn

We report here the case of a term female newborn that developed severe respiratory distress soon after birth. She was found to be a compound heterozygote for both novel mutations in the ABCA3 gene.     

Early stem cell aging in the mature brain

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Survival of salivary gland cancer stem cells requires mTOR signaling

Advanced salivary gland mucoepidermoid carcinoma (MEC) is a relentless cancer that exhibits resistance to conventional chemotherapy. As such, treatment for patients with advanced MEC is tipically radical surgery and radiotherapy. Facial disfigurement and poor quality of life are frequent treatment challenges, and many patients succumb to loco-regional recurrence and/or metastasis. We know that cancer stem-like cells (CSC) drive MEC tumorigenesis. The current study tests the hypothesis that MEC CSC are sensitive to therapeutic inhibition of mTOR. Here, we report a correlation between the long-term clinical outcomes of 17 MEC patients and the intratumoral expression of p-mTOR (p = 0.00294) and p-S6K1 (p = 0.00357). In vitro, we observed that MEC CSC exhibit constitutive activation of the mTOR signaling pathway (i.e., mTOR, AKT, and S6K1), unveiling a potential strategy for targeted ablation of these cells. Using a panel of inhibitors of the mTOR pathway, i.e., rapamycin and temsirolimus (mTOR inhibitors), buparlisib and LY294002 (AKT inhibitors), and PF4708671 (S6K1 inhibitor), we observed consistently dose-dependent decrease in the fraction of CSC, as well as inhibition of secondary sphere formation and self-renewal in three human MEC cell lines (UM-HMC-1,-3A,-3B). Notably, therapeutic inhibition of mTOR with rapamycin or temsirolimus induced preferential apoptosis of CSC, when compared to bulk tumor cells. In contrast, conventional chemotherapeutic drugs (cisplatin, paclitaxel) induced preferential apoptosis of bulk tumor cells and accumulation of CSC. In vivo, therapeutic inhibition of mTOR with temsirolimus caused ablation of CSC and downregulation of Bmi-1 expression (major inducer of stem cell self-renewal) in MEC xenografts. Transplantation of MEC cells genetically silenced for mTOR into immunodeficient mice corroborated the results obtained with temsirolimus. Collectively, these data demonstrated that mTOR signaling is required for CSC survival, and unveiled the therapeutic potential of targeting the mTOR pathway for elimination of highly tumorigenic cancer stem-like cells in salivary gland mucoepidermoid carcinoma.Subject terms: Cancer stem cells, Cancer stem cells, Head and neck cancer, Oral cancer     

Effects of acute intravenous injection of two growth hormone-releasing hormones (GHRH 1-40 and 1-29) on serum growth hormone and other pituitary hormones in short children with pulsatile growth hormone secretion

We administered two different growth hormone-releasing hormones (GHRH) to 20 short, prepubertal children who had spontaneous secretion of growth hormone (GH), assessed from 24-hour GH secretion profiles (72 sampling periods of 20 min). We compared one i.v. injection of 1 microgram/kg of GHRH 1-40 with that of GHRH 1-29 regarding serum concentrations of GH, prolactin, luteinizing hormone, follicle-stimulating hormone and IGF-I. The children were allocated to two groups without statistical randomization. Both groups were given both peptides, with at least 1 week in between. The first group started with GHRH 1-40, the other with GHRH 1-29. The peptides both induced an increased serum concentration of GH of the same magnitude: mean maximal peak of 89 +/- 12 mU/l after GHRH 1-40 and 94 +/- 10 mU/l after GHRH 1-29 (n.s.). The mean difference in maximum serum GH concentration in each child after injection was 52 +/- 9 mU/l, range 1-153 mU/l. GHRH 1-29 also induced a short-term, small increase in the concentrations of prolactin (p less than 0.05), luteinizing hormone (p less than 0.01) and follicle-stimulating hormone (p less than 0.05). We conclude that the shorter sequence GHRH 1-29, when given in a dose of 1 microgram/kg, gives a rise in serum concentration of GH similar to that after the native form GHRH 1-40.