A. Lauwers

Limited redundancy of the proprotein convertase furin in mouse liver

Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver. In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, α5 integrin, lipoprotein receptor-related protein, vitronectin and α1-microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.

Evaluation of the Vitotox™ and RadarScreen assays for the rapid assessment of genotoxicity in the early research phase of drug development

The Vitotox and RadarScreen assays were evaluated as early screens for mutagenicity and clastogenicity, respectively. The Vitotox assay is a bacterial reporter assay in Salmonella typhimurium based on the SOS-response, and it contains a luciferase gene under control of the recN promoter. The RadarScreen assay is a RAD54 promoter-linked beta-galactosidase reporter assay in yeast. The expression of this beta-galactosidase can easily be quantified by use of the substrate d-luciferin-o-beta-galactopyranoside, which is converted into galactose and luciferin that can be measured luminometrically. Recently, an ECVAM workgroup defined a list of 20 genotoxic and 42 non-genotoxic compounds [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108.] that can be used for the validation and/or optimization of in vitro genotoxicity assays. In the present study, this compound set was used for the validation of the assays. Moreover, an additional set of 192 compounds was used to broaden this validation study. The compounds of this additional set can be classified as non-genotoxins and genotoxins and consists of both in-house and reference compounds. In case of the ECVAM compound list, the results from the Vitotox and RadarScreen assays were compared to the genotoxic/non-genotoxic classification of the compounds in this list. In case of the additionally tested compounds, the results of the Vitotox and RadarScreen assays were compared, respectively, with bacterial mutagenicity (Ames) results or in vitro clastogenicity data obtained in-house or from the literature. The validation with respect to the ECVAM compound list resulted in a sensitivity for both the Vitotox and RadarScreen assay of 70% (14/20). If both assays were combined the sensitivity increased to 85% (17/20). Both tests also gave a low number of false positive results. The specificity of the Vitotox and RadarScreen assays was 93% (39/42) and 83% (35/42), respectively. This resulted in a predictivity of the Vitotox and RadarScreen assay of 85% (53/62) and 79% (49/62), respectively. In case both tests were combined the specificity and the predictivity of the Vitotox and RadarScreen assay turned out to be 81% (34/42) and 82% (51/62), respectively. The results from the additional list of 192 compounds confirmed the results found with the ECVAM compound list. The results from the Vitotox assay showed a high correlation with Ames test of 91% (132/145). Subsequently, the RadarScreen assay had a correlation with in vitro clastogenicity of 76% (93/123). The specificity of the Vitotox assay was 94% (90/96) for Ames test results and that of the RadarScreen assay was 74% (34/46) for clastogenicity. Moreover, the sensitivities of the Vitotox and RadarScreen assays were 86% (42/49) and 77% (59/77), respectively. Implementation of the Vitotox and RadarScreen assays in the early research phase of drug development can lead to fast de-selection for genotoxicity. It is expected that this application will reduce the number of compounds that have a positive score in the regulatory Ames and clastogenicity tests. Moreover, problems with a complete compound class can be foreseen at an early time point in the research phase, which gives more time for issue resolution than late detection of these problems with the regulatory tests.

Systematic TLM Measurements of NiSi and PtSi Specific Contact Resistance to n- and p-Type Si in a Broad Doping Range

We present the data on specific silicide-to-silicon contact resistance (rhoc) obtained using optimized transmission-line model structures, processed for a broad range of various n- and p-type Si doping levels, with NiSi and PtSi as the silicides. These structures, despite being attractive candidates for embedding in the CMOS processes, have not been used for NiSi, which is the material of choice in modern technologies. In addition, no database for NiSi-silicon contact resistance exists, particularly for a broad range of doping levels. This letter provides such a database, using PtSi extensively studied earlier as a reference.

Work function of Ni silicide phases on HfSiON and SiO/sub 2/: NiSi, Ni/sub 2/Si, Ni/sub 31/Si/sub 12/, and Ni/sub 3/Si fully silicided gates

A complete determination of the effective work functions (WF) of NiSi, Ni/sub 2/Si, Ni/sub 31/Si/sub 12/ and Ni/sub 3/Si on HfSiON and on SiO/sub 2/ is presented. Conditions for formation of fully silicided (FUSI) gates for NiSi/sub 2/, NiSi, Ni/sub 3/Si/sub 2/, Ni/sub 2/Si, Ni/sub 31/Si/sub 12/ and Ni/sub 3/Si crystalline phases were identified. A double thickness series (HfSiON/SiO/sub 2/) was used to extract WF on HfSiON accounting for charge effects. A strong effect on WF of Ni content is observed for HfSiON, with higher WF for the Ni-rich silicides suggesting unpinning of the Fermi level. A mild dependence is observed for SiO/sub 2/. While all Ni-rich silicides have adequate WF for pMOS applications, Ni/sub 2/Si is most attractive due to its low formation temperature, lower volume expansion and ease of integration. Similar threshold voltages (-0.3 V) were obtained on Ni/sub 2/Si and Ni/sub 31/Si/sub 12/ FUSI HfSiON pMOSFETS.

Mutant Lrp1 Knock-In Mice Generated by Recombinase-Mediated Cassette Exchange Reveal Differential Importance of the NPXY Motifs in the Intracellular Domain of LRP1 for Normal Fetal Development

ABSTRACT Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY→AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR→AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL→AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.

Silicide induced pattern density and orientation dependent transconductance in MOS transistors

For the first time, the influence of the mechanical stress in the transistor channel, induced by the silicide on the source(S)/drain(D) areas, on the transconductance of a MOS transistor has been studied. When scaling down the S/D dimensions, the transconductance can change up to 20% as a result of the increasing stress. This effect is channel orientation dependent. In order to keep the transconductance variation as low as possible, the formation of a low stress silicide is essential.

Self-aligned CoSi/sub 2/ for 0.18 /spl mu/m and below

CoSi/sub 2/ is being used commonly for the advanced IC technologies. There are several process choices to be made for the formation of a high yielding and reproducible silicide. In this paper the various CoSi/sub 2/ technologies are discussed. The scalability of the process of record, the Co/Ti(cap) process are presented for 0.18 /spl mu/m and below.

Scalability of Ni FUSI gate processes: phase and Vt control to 30 nm gate lengths

We demonstrate for the first time the scalability of NiSi and Ni/sub 3/Si FUSI gate processes down to 30 nm gate lengths, with linewidth independent phase and V/sub t/ control. We show that 1-step FUSI is inadequate for NiSi FUSI gates, because it results in incomplete silicidation at low thermal budgets or in a linewidth dependent Ni silicide phase - inducing V/sub t/ shifts - at higher thermal budgets. We show that V/sub t/ and WF shifts are larger on high-K (HfO/sub 2/ (250 mV) or HfSiON (330mV)) than on SiON (110mV) and report Fermi level unpinning for Ni-rich FUSI on high-K. In contrast, we demonstrate the scalability of Ni/sub 3/Si FUSI, with no phase control issues, and report HfSiON Ni/sub 3/Si FUSI PMOS devices with V/sub t/= -0.33 V. Lastly, we show that, for NiSi, phase control down to narrow gate lengths can be obtained with a 2-step FUSI process.

Inactivation of the LRP1 Intracellular NPxYxxL Motif in LDLR-Deficient Mice Enhances Postprandial Dyslipidemia and Atherosclerosis

Objective—The purpose of this study was to determine the significance of the intracellular NPxYxxL motif of LRP1 for the atheroprotective role of this multifunctional receptor. Methods and Results—LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with LDLR-deficient mice, a model for atherosclerosis. In this LDLR−/− background the mutated mice showed a more atherogenic lipoprotein profile, which was associated with a decreased clearance of postprandial lipids because of a compromised endocytosis rate and reduced lipase activity. On an atherogenic diet LRP1 mutant mice revealed a 50% increased development of atherosclerosis. This aggravation was accompanied by an increase in smooth muscle cell (SMC) and collagen content and apoptotic cells in the lesions. The mutation showed, however, a limited impact on basal PDGFR-&bgr; expression and signaling and the antimigratory property of apoE on PDGF-BB–stimulated SMCs. Additionally, levels of LRP1 atherogenic ligands, like MMP2, t-PA, FVIII, and the inflammatory ligand TNF-&agr; showed to be significantly elevated. Conclusion—These findings demonstrate that the NPxYxxL motif is essential for the atheroprotective role of LRP1. This motif is relevant for normal control of lipid metabolism and of atherogenic and inflammatory ligands, but has no pronounced effect on regulating PDGF-BB/PDGFR-&bgr; signaling in SMCs.

Local identification and mapping of the C49 and C54 titanium phases in submicron structures by micro-Raman spectroscopy

In this letter, it is shown that micro-Raman spectroscopy allows easy, nondestructive determination of the C49 and C54 phase of titanium silicide with μm resolution within single structures with area dimensions down to 1×1 μm2 and along isolated line structures with widths down to 0.25 μm. The micro-Raman spectroscopy technique is used to study isolated 0.25–5-μm-wide TiSi2 lines with thicknesses as small as 16 nm that are formed in both crystalline Si and polycrystalline Si. The phase mapping ability of the technique is demonstrated on several 80-μm-long, 0.35-μm-wide TiSi2 lines that are part of four-terminal line resistance devices created using complementary metal–oxide–semiconductor processing.