Investigation of random telegraph signal in two junction layouts of proton irradiated CMOS SPADs.

This paper focuses on the understanding of the Random Telegraph Signal (RTS) in Single-Photon Avalanche Diodes (SPAD). We studied the RTS of two different SPAD layouts, designed and implemented in a 150-nm CMOS process, after proton irradiation. The two structures are characterized by different junction types: the first structure is constituted by a P+/Nwell junction, while the second is formed by a Pwell/Niso junction. RTS occurrence has been measured in about one thousand SPAD pixels and the differences addressed in two layouts are motivated and discussed. Hypotheses on the RTS origin are drawn by analyzing the RTS time constants and the RTS occurrence evolution as a function of the annealing temperature.

Reproducibility of rectal tumor volume delineation using diffusion-weighted MRI: Agreement on volumes between observers.

PURPOSE: To retrospectively evaluate the inter-observer agreement between a radiologist and a radiation oncologist and volume differences, in T2 and diffusion-weighted (DWI) MRI of gross tumor volume (GTV) delineation, in rectal cancer patients. MATERIALS AND METHODS: Two observers, a radiologist and a radiation oncologist, delineated GTVs of 50 patients on T2-weighted MRI (T2GTV) and echo planar DWI (DWIGTV). Observers agreement was assessed using DICE index, Bland-Altman analysis and intra-class correlation coefficient (ICC). Student's t-test was used for GTV comparison. RESULTS: Median T2GTV and DWIGTV were 17.09±14.12 cm3 (1.92-62.03) and 12.79±12.31 cm3 (1.23-62.25) for radiologist, and 16.82±13.66 cm3 (1.78-65.9) and 13.72±12.77 cm3 (1.29-69.75) for radiation oncologist. T2GTV were significantly larger compared to DWIGTV (P<0.001 and P<0.001, for both observers). Mean DICE index for T2GTV and DWIGTV were 0.80±0.07 and 0.77±0.06. The mean difference between the two observers were 0.26cm3 (95% CI: -5.36 to 5.88) and -1.13cm3 (95% CI: -5.70 to 3.44) for T2 and DWI volumes. The ICC for T2 volumes was 0.989 (95% CI: 0.981-0.994) (P<0.001) and 0.992 (95% CI: 0.986-0.996) (P<0.001) for DWI volumes. CONCLUSION: DWI resulted in smaller volumes delineation compared to T2-weighted MRI. Substantial and almost perfect agreements were reported for DWIGTV and T2GTV between radiologist and radiation oncologist. Due to the fact that DWI could be considered a simple technique for volume delineation for radiation oncologist, DWI could be used to improve quality in radiation planning for an accurate boost volume delineation when a dose escalation is investigated.

MONDO: a neutron tracker for particle therapy secondary emission characterisation.

Tumour control is performed in particle therapy using particles and ions, whose high irradiation precision enhances the effectiveness of the treatment, while sparing the healthy tissue surrounding the target volume. Dose range monitoring devices using photons and charged particles produced by the beam interacting with the patient's body have already been proposed, but no attempt has been made yet to exploit the detection of the abundant neutron component. Since neutrons can release a significant dose far away from the tumour region, precise measurements of their flux, production energy and angle distributions are eagerly sought in order to improve the treatment planning system (TPS) software. It will thus be possible to predict not only the normal tissue toxicity in the target region, but also the risk of late complications in the whole body. The aforementioned issues underline the importance of an experimental effort devoted to the precise characterisation of neutron production, aimed at the measurement of their abundance, emission point and production energy. The technical challenges posed by a neutron detector aimed at high detection efficiency and good backtracking precision are addressed within the MONDO (monitor for neutron dose in hadrontherapy) project, whose main goal is to develop a tracking detector that can target fast and ultrafast neutrons. A full reconstruction of two consecutive elastic scattering interactions undergone by the neutrons inside the detector material will be used to measure their energy and direction. The preliminary results of an MC simulation performed using the FLUKA software are presented here, together with the DSiPM (digital SiPM) readout implementation. New detector readout implementations specifically tailored to the MONDO tracker are also discussed, and the neutron detection efficiency attainable with the proposed neutron tracking strategy are reported.

Inhibition of energy metabolism down-regulates the Alzheimer related presenilin 2 gene.

Defects in energy metabolism and oxidative stress play an important role in the pathogenesis of Alzheimer's Disease (AD). In sporadic AD cases, presenilin 2 (PS2) mRNA levels are decreased in brain areas affected by the disease. The aim of the present study was to investigate whether mitochondrial dysfunction might influence PS2 gene expression. We demonstrated that the inhibition of energy metabolism by sodium azide down-regulates PS2 gene expression through modification of promoter activity. No one of the analyzed transcription factors, sensitive to redox status of the cell, could explain this effect. Azide effect on PS2 expression was completely inhibited by the addition of an antioxidant suggesting that the imbalance of the cellular redox homeostasis modulates the expression of this gene.

Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma -secretase processing of the APP and Notch1 transmembrane domains.

A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, gamma-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment gamma (CTFgamma) derivatives are dependent on PS expression and inhibited by a highly selective and potent gamma-secretase inhibitor. Unexpectedly, the APP-CTFgamma derivative is generated by processing between Leu-645 and Val-646 (of APP(695)), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid beta protein peptides. Although the relationship of APP-CTFgamma to the production of amyloid beta protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent gamma-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain.

Detection of the presenilin 1 COOH-terminal fragment in the extracellular compartment: a release enhanced by apoptosis.

Mutations in gene encoding presenilin 1 (PS1) are responsible for the majority of familial Alzheimer's disease (FAD) cases. We studied PS1 localization in HEK293 cells and in primary neurons obtained from rat cortex and hippocampus. We first demonstrated that PS1-CTF, but neither PS1-FL nor PS1-NTF, is released into the medium as a soluble and membrane-associated form. After induction of apoptosis with staurosporine (Sts), we observed a dramatic increase in the level of PS1-CTF in the medium, both in HEK293 and in primary neurons. Immunocytochemical analysis suggested that the release of PS1-CTF might occur via membrane shedding. Abeta(1-42) treatment reduced PS1-CTF extracellular levels. This decrease was strongly associated to an impaired secretion of sAPP fragments, thus suggesting a role of PS1-CTF in the control of trafficking and generation of APP fragments.

Analysis of alpha-2-macroglobulin-2 allele as a risk factor in Alzheimer's disease.

A correlation between a 5-nucleotide deletion polymorphism in the A2M gene and an enhanced risk of developing Alzheimer's disease (AD) was reported. We studied this polymorphism in sporadic AD patients and patients with frontotemporal dementia (FTD) by using an electrophoretical separation of PCR products on a Metaphor gel. Our results did not show any significant difference between A2M-2 allelic frequency (p = 0.89) or genotype frequency (p = 0.97) in the two different clinical series and in control subjects. The frequencies were not significantly different after stratification by APOE epsilon4 status.

Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-amyloid and requires mitogen-activated protein kinase signaling.

Alzheimer's Disease (AD) is characterized by cerebral accumulation of beta-amyloid peptides (Abeta), which are proteolytically derived from beta-amyloid precursor protein (betaAPP). betaAPP metabolism is highly regulated via various signal transduction systems, e.g., several serine/threonine kinases and phosphatases. Several growth factors known to act via receptor tyrosine kinases also have been demonstrated to regulate sbetaAPP secretion. Among these receptors, insulin and insulin-like growth factor-1 receptors are highly expressed in brain, especially in hippocampus and cortex. Emerging evidence indicates that insulin has important functions in brain regions involved in learning and memory. Here we present evidence that insulin significantly reduces intracellular accumulation of Abeta and that it does so by accelerating betaAPP/Abeta trafficking from the trans-Golgi network, a major cellular site for Abeta generation, to the plasma membrane. Furthermore, insulin increases the extracellular level of Abeta both by promoting its secretion and by inhibiting its degradation via insulin-degrading enzyme. The action of insulin on betaAPP metabolism is mediated via a receptor tyrosine kinase/mitogen-activated protein (MAP) kinase kinase pathway. The results suggest cell biological and signal transduction mechanisms by which insulin modulates betaAPP and Abeta trafficking in neuronal cultures.

Presenilin 1 protein directly interacts with Bcl-2.

Presenilin proteins are involved in familial Alzheimer's disease, a neurodegenerative disorder characterized by massive death of neurons. We describe a direct interaction between presenilin 1 (PS1) and Bcl-2, a key factor in the regulation of apoptosis, by yeast two-hybrid interaction system, by co-immunoprecipitation, and by cross-linking experiments. Our data show that PS1 and Bcl-2 assemble into a macromolecular complex, and that they are released from this complex in response to an apoptotic stimulus induced by staurosporine. The results support the idea of cross-talk between these two proteins during apoptosis.

Energy metabolism inhibition impairs amyloid precursor protein secretion from Alzheimer's fibroblasts.

The present study investigates the influence of aglycemia and sodium azide (a Cytochrome c Oxidase inhibitor) on sAPP secretion from skin fibroblasts derived from sporadic AD patients and control subjects. Aglycemia reduced sAPP release in the medium of both AD and control fibroblasts to a similar extent after 2 h incubation. Treatment for 2 h with increasing azide concentrations (1 microM-100 mM) under glucose deprivation did not significantly affect sAPP secretion from control fibroblasts, but was able to significantly inhibit sAPP secretion from AD fibroblasts (maximal inhibition 51%). The failure of antioxidants like glutathione (GSH) or N-acetylcysteine (NAC) to antagonize the azide effect on AD fibroblasts and lipoperoxidation data seemed to rule out the possibility that oxidative stress could mediate the sodium azide effect on sAPP release from AD fibroblasts.

In vitro inhibition by N-acetylcysteine of oxidative DNA modifications detected by 32P postlabeling.

Reactive oxygen species are involved in the pathogenesis of cancer and other chronic degenerative diseases through a variety of mechanisms, including DNA damage. We investigated by 32p and 33P postlabeling analyses the nucleotidic modifications induced in vitro by treating calf thymus DNA with H2O2 and CuSO4, interacting in a Fenton type reaction. Six different enrichment procedures and three chromatographic systems were comparatively assayed. The chromatographic system using phosphate/urea, which is more suitable for detecting bulky DNA adducts, was rather insensitive. In contrast, the system using acetic acid/ammonium formate revealed high levels of mononucleotidic modifications. In terms of ratio of adduct levels in treated and untreated DNA, the enrichment procedures ranked as follows: nuclease P1 (19.6), no enrichment (18.3), digestion to trinucleotides (17.6), digestion to monophosphate mononucleotides (8.4), digestion to dinucleotides (3.4), and extraction with butanol (<1.0). The system using formic acid/ammonium formate was quite efficient in detecting 8-hydroxy-2'-deoxyguanosine. Labeling with 33p further enhanced the sensitivity of the method. The oxidative damage was so intense to produce a strong DNA fragmentation detectable by agarose gel electrophoresis, and nucleotidic modifications were more intense when DNA fragmentation was greater. The DNA alterations produced by H2O2 alone were significantly lower than those produced following reaction of H2O2 with CuSO4. The thiol N-acetylcysteine (NAC) was quite efficient in inhibiting both nucleotidic modifications and DNA fragmentation produced in vitro by either H2O2 or the .OH generating system. These results support at a molecular level the findings of previous studies showing the ability of NAC to inhibit the genotoxicity of peroxides and of reactive oxygen species generated by electron transfer reactions.

Specific role for protein kinase C alpha in the constitutive and regulated secretion of amyloid precursor protein in human skin fibroblasts.

Reduced levels of protein kinase C alpha (PKC alpha) seems to be related to an altered amyloid precursor protein (APP) secretion in fibroblasts from Alzheimer's disease (AD) patients. In this report we used a specific inhibitor of PKC alpha (Gö-6976), to investigate the role of PKC alpha in the basal and phorbol esters regulated secretion of soluble APP (sAPP) in human fibroblasts derived from healthy aged volunteers. Treatment with Gö-6976 alone reduced basal secretion by a maximum of 39%, compared to untreated cells, suggesting the partial dependence of constitutive APP secretory pathway on PKC alpha enzyme. Moreover Gö-6976 treatment completely abolished the effect of phorbol-esters mediated PKC stimulation on sAPP release, suggesting that PKC alpha is the only PKC isoform involved in controlling the secretion of sAPP in human fibroblasts.

Peripheral markers in testing pathophysiological hypotheses and diagnosing Alzheimer's disease.

Alterations in amyloid precursor protein (APP) metabolism, calcium regulation, oxidative metabolism, and transduction systems have been implicated in Alzheimer's disease (AD). Limitations to the use of postmortem brain for examining molecular mechanisms underscore the need to develop a human tissue model representative of the pathophysiological processes that characterize AD. The use of peripheral tissues, particularly of cultured skin fibroblasts derived from AD patients, could complement studies of autopsy samples and provide a useful tool with which to investigate such dynamic processes as signal transduction systems, ionic homeostasis, oxidative metabolism, and APP processing. Peripheral cells as well as body fluids (i.e., plasma and CSF) could also provide peripheral biological markers for the diagnosis of AD. The criteria required for a definite diagnosis of AD presently include clinical criteria in association with histopathologic evidence obtained from biopsy or autopsy. Thus, the use of peripheral markers as a diagnostic tool, either to predict or at least to confirm a diagnosis, may be of great importance.

Oxidative metabolism in cultured fibroblasts derived from sporadic Alzheimer's disease (AD) patients.

Fibroblasts from Alzheimer's disease (AD) patients displayed decreased cytochrome c oxidase (complex IV) activity (P < 0.05). The basal oxygen consumption rate (QO2) and the response to an uncoupler of oxidative phosphorylation did not differ between AD and control fibroblasts. The QO2 of AD fibroblasts was more susceptible (P < 0.05) to inhibition by azide in the range 0.5-5 mM. The basal intracellular pH (pHi) in AD fibroblasts was significantly more acidic than in control ones. The results support the hypothesis that subtle dysfunctions of oxidative energy-producing processes are present in fibroblasts from sporadic AD patients. The alterations observed scantly influence the fibroblasts functioning even in stressful conditions; however in tissues, such as the brain, that rely heavily on oxidative metabolism for their function, similar alterations may trigger molecular mechanisms leading to cell damage.

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells.

The present study investigates the influence of energy related metabolic stress on amyloid precursor protein (APP) non-amyloidogenic secretory processing in COS cells. The effect of glucose deprivation on soluble APP (sAPP) secretion has been evaluated: incubation of COS cells with 50 mM 2-deoxy-D-glucose (2-DG) in glucose free medium was able to reduce sAPP secretion (-26%). Sodium azide (NaN3), an inhibitor of cytochrome c oxidase (complex IV of the mitochondrial electron transfer chain) decreased sAPP release in a concentration dependent way (maximum -75%). Treatment of COS cells with the antioxidant glutathione (GSH) fully antagonized the inhibitory effect of azide (1 mM) and elicited sAPP release over basal level. These results suggest that the inhibition of energy metabolism can influence APP processing leading to a decreased secretion of non-amyloidogenic fragments of APP.

Fibroblasts of patients affected by Down's syndrome oversecrete amyloid precursor protein and are hyporesponsive to protein kinase C stimulation.

The present study investigates the ability of the pharmacologic activation of protein kinase C (PKC) to modulate amyloid precursor protein (APP) secretion in human skin fibroblasts from patients affected by Down's syndrome (DS). We assessed DS subjects at the Hospital Institute of Sospiro, Cremona, and at the Alzheimer's Disease Unit of the Sacred Heart Hospital in Brescia, and we subdivided them into nondemented (NDS) and demented (DDS) patients. All DS patients were trisomy 21 karyotype. DS fibroblasts had an increased content of APP immunoreactive material as revealed by immunocytochemistry analysis. The basal secretion of soluble APP was higher (+94.6%) in Down's cells with respect to controls. The observation on the fibroblasts prepared from DS is consistent with these patients' possessing an extra copy of the APP gene (mapped on chromosome 21) leading to increased APP expression. Phorbol-12,13-dibutyrate (PdBu, 9 to 150 nM) treatment promoted a dose-dependent increase of secreted APP in the conditioned medium of control fibroblasts. The peak response (+102.2%) was attained using 150 nM PdBu. In Down's fibroblasts, PdBu stimulated APP secretion already maximally at low concentrations (9 nM), but the peak response, due to the higher basal release, was lower on a percentage basis (+16.4%) than in control fibroblasts. The results indicate that in Down's fibroblasts the mechanisms controlling APP release are at least quantitatively altered. In addition, these results suggest caution when using information obtained from Down's patients to model Alzheimer's disease biochemical defects.

Peripheral cells as an investigational tool for Alzheimer's disease.

A number of abnormalities in metabolic and biochemical processes have been found in cultured skin fibroblasts derived from patients affected by Alzheimer's disease (AD). An example of the successful use of peripheral cells to examine a cell biological abnormality in AD are the studies on transduction systems and on APP metabolism, mostly performed on fibroblasts from AD donors. In fact, some of the described alterations mirror events that have also been demonstrated to occur in the AD brain. Within this context data obtained using peripheral cells may help to identify and to test hypotheses on the primary pathophysiological mechanisms leading to AD. In perspective, the identification of peripheral biological markers could provide a useful aid in AD and could allow identification of stages of of the disease or subgrouping of patients, possibly helping to predict the response to treatment.