BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues

sox9b Is a Key Regulator of Pancreaticobiliary Ductal System Development

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies

Resequencing PNMT in European hypertensive and normotensive individuals: no common susceptibilily variants for hypertension and purifying selection on intron 1

<p>Abstract</p> <p>Background</p> <p>Human linkage and animal QTL studies have indicated the contribution of genes on Chr17 into blood pressure regulation. One candidate gene is <it>PNMT</it>, coding for phenylethanolamine-N-methyltransferase, catalyzing the synthesis of epinephrine from norepinephrine.</p> <p>Methods</p> <p>Fine-scale variation of <it>PNMT </it>was screened by resequencing hypertensive (n = 50) and normotensive (n = 50) individuals from two European populations (Estonians and Czechs). The resulting polymorphism data were analyzed by statistical genetics methods using Genepop 3.4, PHASE 2.1 and DnaSP 4.0 software programs. <it>In silico </it>prediction of transcription factor binding sites for intron 1 was performed with MatInspector 2.2 software.</p> <p>Results</p> <p><it>PNMT </it>was characterized by minimum variation and excess of rare SNPs in both normo- and hypertensive individuals. None of the SNPs showed significant differences in allelic frequencies among population samples, as well as between screened hypertensives and normotensives. In the joint case-control analysis of the Estonian and the Czech samples, hypertension patients had a significant excess of heterozygotes for two promoter region polymorphisms (SNP-184; SNP-390). The identified variation pattern of <it>PNMT </it>reflects the effect of purifying selection consistent with an important role of PNMT-synthesized epinephrine in the regulation of cardiovascular and metabolic functions, and as a CNS neurotransmitter. A striking feature is the lack of intronic variation. <it>In silico </it>analysis of <it>PNMT </it>intron 1 confirmed the presence of a human-specific putative Glucocorticoid Responsive Element (GRE), inserted by <it>Alu</it>-mediated transfer. Further analysis of intron 1 supported the possible existence of a full Glucocorticoid Responsive Unit (GRU) predicted to consist of multiple gene regulatory elements known to cooperate with GRE in driving transcription. The role of these elements in regulating <it>PNMT </it>expression patterns and thus determining the dynamics of the synthesis of epinephrine is still to be studied.</p> <p>Conclusion</p> <p>We suggest that the differences in PNMT expression between normotensives and hypertensives are not determined by the polymorphisms in this gene, but rather by the interplay of gene expression regulators, which may vary among individuals. Understanding the determinants of PNMT expression may assist in developing PNMT inhibitors as potential novel therapeutics.</p

The Chromatin Modifier MSK1/2 Suppresses Endocrine Cell Fates during Mouse Pancreatic Development

Type I diabetes is caused by loss of insulin-secreting beta cells. To identify novel, pharmacologically-targetable histone-modifying proteins that enhance beta cell production from pancreatic progenitors, we performed a screen for histone modifications induced by signal transduction pathways at key pancreatic genes. The screen led us to investigate the temporal dynamics of ser-28 phosphorylated histone H3 (H3S28ph) and its upstream kinases, MSK1 and MSK2 (MSK1/2). H3S28ph and MSK1/2 were enriched at the key endocrine and acinar promoters in E12.5 multipotent pancreatic progenitors. Pharmacological inhibition of MSK1/2 in embryonic pancreatic explants promoted the specification of endocrine fates, including the beta-cell lineage, while depleting acinar fates. Germline knockout of both Msk isoforms caused enhancement of alpha cells and a reduction in acinar differentiation, while monoallelic loss of Msk1 promoted beta cell mass. Our screen of chromatin state dynamics can be applied to other developmental contexts to reveal new pathways and approaches to modulate cell fates

Plasma-assisted ALD for the conformal deposition of SiO2 : process, material and electronic properties

Plasma-assisted atomic layer deposition (ALD) was used to deposit SiO2 films in the temperature range of Tdep = 50–400°C on Si(100). H2Si[N(C2H5)2]2 and an O2 plasma were used as Si precursor and oxidant, respectively. The ALD growth process and material properties were characterized in detail. Ultrashort precursor doses (~50 ms) were found to be sufficient to reach self-limiting ALD growth with a growth-per-cycle of ~1.2 Å (Tdep = ~200°C) leading to SiO2 films with O/Si ratio of ~2.1. Moreover, the plasma ALD process led to a high conformality (95–100%) for trenches with aspect ratios of ~30. In addition, the electronic (interface) properties of ultrathin ALD SiO2 films and ALD SiO2/Al2O3 stacks were studied by capacitance-voltage and photoconductance decay measurements. The interface quality associated with SiO2 was improved significantly by using an ultrathin ALD Al2O3 capping layer and annealing. The interface defect densities decreased from ~1×1012 eV-1 cm-2 (at mid gap) for single layer SiO2 to &lt;1011 eV-1 cm-2 for the stacks. Correspondingly, ultralow surface recombination velocities &lt;3 cm/s were obtained for n-type Si. The density and polarity of the fixed charges associated with the stacks were found to be critically dependent on the SiO2 thickness (1–30 nm)

Loss of Smad4 function in pancreatic tumors: C-terminal truncation leads to decreased stability.

At early stages of tumorigenesis, the transforming growth factor-beta (TGF-beta) signaling pathway is thought to have tumor suppressor activity as a result of its ability to arrest the growth of epithelial cells. Smad4 plays a pivotal role in the TGF-beta signaling pathway and has been identified as a tumor suppressor, being mutated or deleted in approximately 50% of pancreatic carcinomas and 15% of colorectal cancers. A nonsense mutation generating a C-terminal truncation of 38 amino acids in the Smad4 protein has been identified in a pancreatic adenocarcinoma (Hahn, S. A., Schutte, M., Hoque, A. T., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E. (1996) Science 271, 350-353), and here we investigate the functional consequences of this mutation. We demonstrate that the C-terminal truncation prevents Smad4 homomeric complex formation and heteromeric complex formation with activated Smad2. Furthermore, the mutant protein is unable to be recruited to DNA by transcription factors and hence cannot form transcriptionally active DNA-binding complexes. These observations are supported by molecular modeling, which indicates that the truncation removes residues critical for homomeric and heteromeric Smad complex formation. We go on to show that the mutant Smad4 is highly unstable compared with wild type Smad4 and is rapidly degraded through the ubiquitin-proteasome pathway. Consistent with this, we demonstrate that the pancreatic adenocarcinoma harboring this mutated allele, in conjunction with loss of the other allele, expresses no Smad4 protein. Thus we conclude that these tumors completely lack Smad4 activity

Influence of the Oxidant on the Chemical and Field-Effect Passivation of Si by ALD Al2O3

Differences in Si surface passivation by aluminum oxide (Al2O3) films synthesized using H2O and O-3-based thermal atomic layer deposition (ALD) and plasma ALD have been revealed. A low interface defect density of D-it = similar to 1011 eV(-1) cm(-2) was obtained after annealing, independent of the oxidant. This low D-it was found to be vital for the passivation performance. Field-effect passivation was less prominent for H2O-based ALD Al2O3 before and after annealing, whereas for as-deposited ALD films with an O-2 plasma or O-3 as the oxidants, the field-effect passivation was impaired by a very high Dit. (C) 2010 The Electrochemical Society

Influence of the oxidant on the chemical and field-effect passivation of Si by ALD Al2O3

Differences in Si surface passivation by aluminum oxide (Al2O3) films synthesized using H2O and O3-based thermal atomic layer deposition (ALD) and plasma ALD have been revealed. A low interface defect density of Dit=~1011 eV-1 cm-2 was obtained after annealing, independent of the oxidant. This low Dit was found to be vital for the passivation performance. Field-effect passivation was less prominent for H2O-based ALD Al2O3 before and after annealing, whereas for as-deposited ALD films with an O2 plasma or O3 as the oxidants, the field-effect passivation was impaired by a very high Dit

Influence of the Oxidant on the Chemical and Field-Effect Passivation of Si by ALD Al2O3

Differences in Si surface passivation by aluminum oxide (Al2O3) films synthesized using H2O and O-3-based thermal atomic layer deposition (ALD) and plasma ALD have been revealed. A low interface defect density of D-it = similar to 1011 eV(-1) cm(-2) was obtained after annealing, independent of the oxidant. This low D-it was found to be vital for the passivation performance. Field-effect passivation was less prominent for H2O-based ALD Al2O3 before and after annealing, whereas for as-deposited ALD films with an O-2 plasma or O-3 as the oxidants, the field-effect passivation was impaired by a very high Dit. (C) 2010 The Electrochemical Society