Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

PMCID: PMC3553106This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Increased expression of endothelial lipase in symptomatic and unstable carotid plaques

The aim of this study was to evaluate endothelial lipase (EL) protein expression in advanced human carotid artery plaques (HCAP) with regard to plaque (in)stability and the incidence of symptoms. HCAP were collected from 66 patients undergoing carotid endarterectomy (CEA). The degree of plaque (in)stability was estimated by ultrasound and histology. In HCAP sections, EL expression was determined by immunostaining and the intensity was assessed on a semi-quantitative scale (low: <25%, high: >25% positive cells). Monocytes and macrophages in adjacent HCAP sections were stained with a CD163 specific antibody. High EL staining was more prevalent in histologically unstable plaques (in 33.3% of fibrous plaques, 50% of ulcerated non-complicated plaques and 79.2% of ulcerated complicated plaques; χ2 test, p = 0.004) and in the symptomatic group (70.8 vs. 42.9% in the asymptomatic group; χ2 test, p = 0.028). The majority of EL immunostaining was found in those HCAP regions exhibiting a strong CD163 immunostaining. EL in HCAP might be a marker and/or promoter of plaque instability and HCAP-related symptomatology

Cystatin C Deficiency Promotes Epidermal Dysplasia in K14-HPV16 Transgenic Mice

Cysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors.We tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice.Enhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization

Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics

Oncogenic mutations in the mitogen activated protein kinase (MAPK) pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2). We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells

Cathepsin K Null Mice Show Reduced Adiposity during the Rapid Accumulation of Fat Stores

Growing evidences indicate that proteases are implicated in adipogenesis and in the onset of obesity. We previously reported that the cysteine protease cathepsin K (ctsk) is overexpressed in the white adipose tissue (WAT) of obese individuals. We herein characterized the WAT and the metabolic phenotype of ctsk deficient animals (ctsk−/−). When the growth rate of ctsk−/− was compared to that of the wild type animals (WT), we could establish a time window (5–8 weeks of age) within which ctsk−/−display significantly lower body weight and WAT size as compared to WT. Such a difference was not observable in older mice. Upon treatment with high fat diet (HFD) for 12 weeks ctsk−/− gained significantly less weight than WT and showed reduced brown adipose tissue, liver mass and a lower percentage of body fat. Plasma triglycerides, cholesterol and leptin were significantly lower in HFD-fed-ctsk−/− as compared to HFD-fed WT animals. Adipocyte lipolysis rates were increased in both young and HFD-fed-ctsk−/−, as compared to WT. Carnitine palmitoyl transferase-1 activity, was higher in mitochondria isolated from the WAT of HFD treated ctsk−/− as compared to WT. Together, these data indicate that ctsk ablation in mice results in reduced body fat content under conditions requiring a rapid accumulation of fat stores. This observation could be partly explained by an increased release and/or utilization of FFA and by an augmented ratio of lipolysis/lipogenesis. These results also demonstrate that under a HFD, ctsk deficiency confers a partial resistance to the development of dyslipidemia

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively

Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon

TGF-β in progression of liver disease

Transforming growth factor-β (TGF-β) is a central regulator in chronic liver disease contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver-damage-induced levels of active TGF-β enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Being recognised as a major profibrogenic cytokine, the targeting of the TGF-β signalling pathway has been explored with respect to the inhibition of liver disease progression. Whereas interference with TGF-β signalling in various short-term animal models has provided promising results, liver disease progression in humans is a process of decades with different phases in which TGF-β or its targeting might have both beneficial and adverse outcomes. Based on recent literature, we summarise the cell-type-directed double-edged role of TGF-β in various liver disease stages. We emphasise that, in order to achieve therapeutic effects, we need to target TGF-β signalling in the right cell type at the right time

Distinct clinical and neuropathological features of G51D SNCA mutation cases compared with SNCA duplication and H50Q mutation

Background: We and others have described the neurodegenerative disorder caused by G51D SNCA mutation which shares characteristics of Parkinson’s disease (PD) and multiple system atrophy (MSA). The objective of this investigation was to extend the description of the clinical and neuropathological hallmarks of G51D mutant SNCA-associated disease by the study of two additional cases from a further G51D SNCA kindred and to compare the features of this group with a SNCA duplication case and a H50Q SNCA mutation case. Results: All three G51D patients were clinically characterised by parkinsonism, dementia, visual hallucinations, autonomic dysfunction and pyramidal signs with variable age at disease onset and levodopa response. The H50Q SNCA mutation case had a clinical picture that mimicked late-onset idiopathic PD with a good and sustained levodopa response. The SNCA duplication case presented with a clinical phenotype of frontotemporal dementia with marked behavioural changes, pyramidal signs, postural hypotension and transiently levodopa responsive parkinsonism. Detailed post-mortem neuropathological analysis was performed in all cases. All three G51D cases had abundant α-synuclein pathology with characteristics of both PD and MSA. These included widespread cortical and subcortical neuronal α-synuclein inclusions together with small numbers of inclusions resembling glial cytoplasmic inclusions (GCIs) in oligodendrocytes. In contrast the H50Q and SNCA duplication cases, had α-synuclein pathology resembling idiopathic PD without GCIs. Phosphorylated α-synuclein was present in all inclusions types in G51D cases but was more restricted in SNCA duplication and H50Q mutation. Inclusions were also immunoreactive for the 5G4 antibody indicating their highly aggregated and likely fibrillar state. Conclusions: Our characterisation of the clinical and neuropathological features of the present small series of G51D SNCA mutation cases should aid the recognition of this clinico-pathological entity. The neuropathological features of these cases consistently share characteristics of PD and MSA and are distinct from PD patients carrying the H50Q or SNCA duplication

Geodesy and metrology with a transportable optical clock

partially_open24openGrotti, Jacopo; Koller, Silvio; Vogt, Stefan; Häfner, Sebastian; Sterr, Uwe; Lisdat, Christian; Denker, Heiner; Voigt, Christian; Timmen, Ludger; Rolland, Antoine; Baynes, Fred N.; Margolis, Helen S.; Zampaolo, Michel; Thoumany, Pierre; Pizzocaro, Marco; Rauf, Benjamin; Bregolin, Filippo; Tampellini, Anna; Barbieri, Piero; Zucco, Massimo; Costanzo, Giovanni A.; Clivati, Cecilia; Levi, Filippo; Calonico, DavideGrotti, Jacopo; Koller, Silvio; Vogt, Stefan; Häfner, Sebastian; Sterr, Uwe; Lisdat, Christian; Denker, Heiner; Voigt, Christian; Timmen, Ludger; Rolland, Antoine; Baynes, Fred N.; Margolis, Helen S.; Zampaolo, Michel; Thoumany, Pierre; Pizzocaro, Marco; Rauf, Benjamin; Bregolin, Filippo; Tampellini, Anna; Barbieri, Piero; Zucco, Massimo; Costanzo, Giovanni A.; Clivati, Cecilia; Levi, Filippo; Calonico, David