Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Body mass growth in common marmosets: Toward a model of pediatric obesity

While much is known about adult obesity in nonhuman primates, very little is known regarding development of childhood adiposity. As small monkeys that are easy to handle and have a relatively fast life history, common marmoset monkeys (Callithrix jacchus) offer interesting opportunities to examine the question of fat versus lean mass growth in a nonhuman primate. This article provides an overview of our understanding of early life growth in mass in marmoset monkeys, based primarily upon our past 20 years of research, culminating in our recent findings on early life obesity in this species. Common marmosets display variance in early life growth patterns that is related to both pre‐ and postnatal factors and the marmoset uterine environment is exquisitely designed to reflect resources available for the gestation of multiple offspring, making them an interesting model of developmental programming. We have demonstrated that obesity can be generated in very early life in captive marmosets, with excess adiposity evident by one month of age, making this species a potentially valuable model in which to study pediatric obesity and its sequelae. Birth weight is associated with adiposity in animals vulnerable to obesity. Early life exposure to higher fat diets enhances the chances of postweaning obesity development. However, overall higher food consumption is also associated with obesity development at later ages. One unexpected finding in our studies has been the relatively high body fat percentage of neonatal (12–18%) marmosets suggesting that hypotheses regarding the uniqueness of high human neonatal adiposity merit further examination. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors

Herein, we report the synthesis and structure–activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC₅₀ = 0.8 μM).     

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs)

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX₁R/OX₂R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.     

Synthesis and evaluation of Janus type nucleosides as potential HCV NS5B polymerase inhibitors

The synthesis of new ribo and 2′-β-C-methyl ribo Janus type nucleosides J-AA, J-AG and J-AU is reported along with their ability to block HCV and HIV replication. Their toxicity was also assessed in Huh7, human lymphocytes, CEM and Vero cells.     

TGF-b Superfamily Cytokine MIC-1/GDF15 Is a Physiological Appetite and Body Weight Regulator

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1^−/−) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1^−/− mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1^−/− mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.