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In mammalian prion diseases, an abnormally folded, aggregated form of the prion protein (PrP(Sc)) appears to catalyze a conformational switch of its cellular isoform (PrP(C)) to an aggregated state. A similar prion-like phenomenon has been reported for the Saccharomyces cerevisiae translation termination factor Sup35p that can adopt a self-propagating conformation. We have compared aggregation propensities of chimeric proteins derived from the Sup35p prion domain NM and PrP in vitro and in the cytosol of mammalian cells. Sup35p-NM and PrP displayed strikingly different aggregation behaviors when expressed in mammalian cells, with NM remaining soluble and cytosolic PrP spontaneously aggregating due to the globular domain of PrP. When fused to PrP(90-230), Sup35p-M exhibited an inhibitory effect for nucleation but increased aggregate growth, potentially by facilitating recruitment of newly synthesized chimeric proteins into the growing aggregates. This effect, however, could, to some extent, be counteracted by the prion-forming region Sup35p-N, thereby increasing aggregate frequency. Interestingly, a lowered nucleation rate was also observed in the presence of the amino-terminal region of PrP, suggesting that Sup35p-M and PrP(23-90) share some biological function in prion protein assembly. Our results provide new insights into prion protein aggregation behaviors, demonstrating the impact of dynamic interactions between prion domains and suggesting that aggregation of yeast and mammalian prion proteins is strongly influenced by yet unidentified cellular conditions or factors.

Prion diseases are fatal neurodegenerative infectious disorders for which no therapeutic or prophylactic regimens exist. Our work aims to eliminate PrP(c) as substrate for the conversion into PrP(Sc) and to increase the cellular clearance capacity of PrP(Sc). In order to achieve the first objective, we used chemical compounds which interfere with the subcellular trafficking of PrP(c), e.g. by intracellular re-routing. Recently, we found that PrP(c) requires cholesterol for cell surface localisation. Treatment with mevinolin significantly reduced the amount of cell surface PrP(c) and led to its accumulation in the Golgi compartment. These data show that cholesterol is essential for the cell surface localisation of PrP(c), which is in turn known to be necessary for the formation of PrP(Sc). Another anti-prion strategy uses RNA and peptide aptamers directed against PrP(c). We have selected peptide aptamers using a constrained peptide library presented on the active site loop of the Escherichia coli protein TrxA in a Y2H screen. Several peptides reproducibly binding to PrP(c) in several assays were identified. Preliminary data indicate that selected peptide aptamers are able to interfere with prion propagation in prion-infected cells. To obtain additive effects we have tried to clarify cellular mechanisms that enable cells to clear prion infectivity. This goal was achieved by selective interference in intracellular signalling pathways which apparently also increase the cellular autophagy machinery. Finally, we have tried to establish an active auto-vaccination approach directed against PrP, which gave some positive preliminary results in the mouse system. This might open the door to classical immunological interference techniques.

Prion diseases are rare and obligatory fatal neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrPSc) of the host-encoded prion protein (PrPc). Prophylactic and therapeutic regimens against prion diseases are very limited. To extend such strategies we selected peptide aptamers binding to PrP from a combinatorial peptide library presented on the Escherichia coli thioredoxin A (trxA) protein as a scaffold. In a yeast two-hybrid screen employing full-length murine PrP (aa 23-231) as a bait we identified three peptide aptamers that reproducibly bind to PrP. Treatment of prion-infected cells with recombinantly expressed aptamers added to the culture medium abolished PrPSc conversion with an IC50 between 350 and 700 nM. For expression in eukaryotic cells, peptide aptamers were fused to an N-terminal signal peptide for entry of the secretory pathway. The C terminus was modified by a glycosyl-phosphatidyl-inositol-(GPI) anchoring signal, a KDEL retention motif and the transmembrane and cytosolic domain of LAMP-I, respectively. These peptide aptamers retained their binding properties to PrPc and, depending on peptide sequence and C-terminal modification, interfered with endogenous PrPSc conversion upon expression in prion-infected cells. Notably, infection of cell cultures could be prevented by expression of KDEL peptide aptamers. For the first time, we show that trxA-based peptide aptamers can be targeted to the secretory pathway, thereby not losing the affinity for their target protein. Beside their inhibitory effect on prion conversion, these molecules could be used as fundament for rational drug design.

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.

The fear of malevolent use of variola virus by terrorists has led to the implementation of a health care worker vaccination program and to the consideration of vaccination for the general public. However, due to concerns about side effects of the classical smallpox vaccine, especially for immunocompromised individuals, a safer vaccine is urgently needed. We characterized the immunogenicity of modified vaccinia virus Ankara (MVA), one of the more promising alternative smallpox vaccines, in a cohort of 10 chronically HIV-1-infected individuals undergoing highly active antiretroviral therapy (HAART). Nine subjects received smallpox vaccination as children while one subject was never vaccinated against smallpox. All the subjects had CD4 counts >400 cells/mm(3) and 8 out of 10 had undetectable viral loads. MVA was able to elicit humoral and cellular immune responses in the majority of individuals. Vaccinia-specific antibodies were mainly of the IgG class while T cells specific to vaccinia were predominantly CD8(+). The immune responses were maintained over 1 year. Similar vaccinia specific humoral immune responses were observed when our cohort of HIV-1-infected individuals was compared to smallpox-vaccinated healthy subjects. The observed immune responses suggest that the highly attenuated MVA could be used as a substitute vaccine against smallpox in chronically HIV-1-infected individuals undergoing HAART.

The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.

Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrP(Sc)). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrP(C)) has been reported to abolish PrP(Sc) infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrP(C), active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP-virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrP(C) in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrP(Sc) in prion-infected cells. If also effective in vivo, PrP-virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases.

1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP(Sc) propagation in prion-infected cells.2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP(Sc)) and normal prion protein (PrP(c)), respectively. Sera positive in all tests were chosen for PrP(Sc) inhibition studies in cell culture.3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95-123 led to a transient reduction of PrP(Sc) levels in persistently prion-infected cells.4. Thus, anti-PrP antibodies interfering with PrP(Sc) propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95-123 for the treatment of prion diseases.

The neuronal perturbations in Alzheimer’s disease are attributed to the formation of extracellular amyloid-β (Aβ) neuritic plaques, composed predominantly of the neurotoxic Aβ42 isoform. Although the plaques have demonstrated a role in synaptic dysfunction, neuronal cytotoxicity has been attributed to soluble Aβ42 oligomers. The 37kDa/67kDa laminin receptor has been implicated in Aβ42 shedding and Aβ42-induced neuronal cytotoxicity, as well as internalization of this neurotoxic peptide. As the cellular prion protein binds to both LRP/LR and Aβ42, the mechanism underlying this cytotoxicity may be indirectly due to the PrPc-Aβ42 interaction with LRP/LR. The effects of this interaction were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assays. PrPc overexpression significantly enhanced Aβ42 cytotoxicity in vitro, while PrP–/– cells were more resistant to the cytotoxic effects of Aβ42 and exhibited significantly less cell death than PrPc expressing N2a cells. Although anti-LRP/LR specific antibody IgG1-iS18 significantly enhanced cell viability in both pSFV1-huPrP1-253 transfected and non-transfected cells treated with exogenous Aβ42, it failed to have any cell rescuing effect in PrP–/– HpL3-4 cells. These results suggest that LRP/LR plays a significant role in Aβ42-PrPc mediated cytotoxicity and that anti-LRP/LR specific antibodies may serve as potential therapeutic tools for Alzheimer’s disease.

Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrP Sc . Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrP Sc uptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independently of the prion strain and reduced PrP Sc formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrP Sc formation independent of the prion strain. IMPORTANCE Recently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro . GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.

Prions as causative agents of transmissible spongiform encephalopathies have been well investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors, and the interaction of prions with cellular components. We investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) were examined. Both cell lines can be persistently infected with mouse adapted prion strains, i.e., RML. Using a mammalian retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected, and anti-prion compound-treated murine neuronal cell lines, including clonal cell populations. The results suggest that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and responsive to anti-prion compound treatment.

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.

The cellular prion protein PrP(c) is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI-) anchor and is localized in lipid rafts, membrane microdomains characterized by a high content of sphingolipids and cholesterol. Previous studies revealed that perturbation of cholesterol synthesis prevents prion conversion, explained by redistribution of PrP(c) at the plasma membrane. We investigated the influence of inhibition of cholesterol synthesis by the HMG-CoA-reductase inhibitor mevinolin on the trafficking of PrP(c) in neuronal cells. Treatment with mevinolin significantly reduces the amount of surface PrP(c) and leads to its accumulation in the Golgi compartment. Analysis of mutant PrPs highlights the importance of the GPI-anchor for raft localization and provides information about domains implicated in lipid raft association of PrP in the secretory pathway. Our data show that cholesterol is essential for the cell surface localization of PrP(c), known to be necessary for prion conversion.

Due to German regulations, acceptance and consistency tests have to be obtained by 12.31.2005 for all equipment used for computed radiography according to special standards published in DIN 6868. This article familiarizes all users with the most important aspects of these standards. In addition, explanatory and background information for establishing these regulations are provided.

In a survey including all mammography units in Bavaria, entrance doses in air and other quality-relevant parameters were measured with standard settings and under comparable conditions. The study was aimed to discover discrepancies between practical mammographic imaging techniques and the demands established in the European Guidelines. Test images were made by the operators themselves with phantoms used for constancy checks. Entrance dose values were measured with official film dosimeters. The survey showed that more than 95 % of all mammographic units in Bavaria comply with the European guidelines with respect to radiation exposure and imaging sharpness.

Prion diseases are fatal transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We have shown previously that the chemical compound suramin induced aggregation of fully matured PrP(c) in post-ER compartments, thereby, activating a post-ER quality control mechanism and preventing cell surface localization of PrP by intracellular re-routing of aggregated PrP from the Golgi/TGN directly to lysosomes. Of note, drug-induced PrP aggregates were not toxic and could easily be degraded by neuronal cells. Here, we focused on determining the PrP domains mediating these effects. Using PrP deletion mutants we show that intracellular re-routing but not aggregation depends on the N-terminal PrP (aa 23-90) and, more precisely, on the preoctarepeat domain (aa 23-50). Fusion of the PrP N-terminus to the GPI-anchored protein Thy-1 did not cause aggregation or re-routing of the chimeric protein, indicating that the N-terminus is only active in re-routing when prion protein aggregation occurs. Insertion of a region with a comparable primary structure contained in the PrP paralogue prnd/doppel (aa 27-50) into N-terminally deleted PrP re-established the re-routing phenotype. Our data reveal an important role for the conserved preoctarepeat region of PrP, namely controlling the intracellular trafficking of misfolded PrP.

Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.

Prion diseases are neurodegenerative infectious disorders for which no prophylactic regimens are known. In order to induce antibodies/auto-antibodies directed against surface-located PrP(c), we used a covalently linked dimer of mouse prion protein expressed recombinantly in Escherichia coli. Employing dimeric PrP as an immunogen we were able to effectively overcome autotolerance against murine PrP in PrP wild-type mice without inducing obvious side effects. Treatment of prion-infected mouse cells with polyclonal anti-PrP antibodies generated in rabbit or auto-antibodies produced in mice significantly inhibited endogenous PrP(Sc) synthesis. We show that polyclonal antibodies are binding to surface-located PrP(c), thereby interfering with prion biogenesis. This effect is much more pronounced in the presence of full IgG molecules, which, unlike Fab fragments, seem to induce a significant cross-linking of surface PrP. In addition, we found immune responses against different epitopes when comparing antibodies induced in rabbits and PrP wild-type mice. Only in the auto-antibody situation in mice an immune reaction against a region of PrP is found that was reported to be involved in the PrP(Sc) conversion process. Our data point to the possibility of developing means for an active immunoprophylaxis against prion diseases.

Chronic wasting disease (CWD) is a fatal transmissible spongiform encephalopathy affecting white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose (Alces alces shirasi) in North America. In southeastern Wyoming average annual CWD prevalence in mule deer exceeds 20% and appears to contribute to regional population declines. We determined the effect of CWD on mule deer demography using age-specific, female-only, CWD transition matrix models to estimate the population growth rate (λ). Mule deer were captured from 2010–2014 in southern Converse County Wyoming, USA. Captured adult (≥ 1.5 years old) deer were tested ante-mortem for CWD using tonsil biopsies and monitored using radio telemetry. Mean annual survival rates of CWD-negative and CWD-positive deer were 0.76 and 0.32, respectively. Pregnancy and fawn recruitment were not observed to be influenced by CWD. We estimated λ = 0.79, indicating an annual population decline of 21% under current CWD prevalence levels. A model derived from the demography of only CWD-negative individuals yielded; λ = 1.00, indicating a stable population if CWD were absent. These findings support CWD as a significant contributor to mule deer population decline. Chronic wasting disease is difficult or impossible to eradicate with current tools, given significant environmental contamination, and at present our best recommendation for control of this disease is to minimize spread to new areas and naïve cervid populations.

Here we introduce a protocol for Triton X-114 extraction which we used in our recently-published paper (Taguchi et al., 2013). It is a versatile method to concentrate or partially purify hydrophobic proteins. The presented protocol is based on the protocol published by Bordier (Bordier, 1981) but more simplified and down-scaled for more small-scale and simpler use (Taguchi et al., 2013). Triton X-114 (TX114) is a non-ionic detergent which has a relatively low clouding point at 22 °C and separates into detergent (Det) and aqueous (Aq) phase at temperatures above the clouding point. During phase separation, hydrophobic solutes in the TX114 solution are sequestered to the Det phase, while hydrophilic solutes are sequestered to the Aq phase. Utilizing this phenomenon, TX114 extraction is a very versatile technique to efficiently concentrate hydrophobic proteins, especially glycosylphosphatidylinositol (GPI)-anchored proteins like the prion protein (PrP), because they have substantial amounts of highly hydrophobic moieties. Besides, phase separation using TX114 tolerates a variety of conditions, e.g. different pH or relatively low concentrations of guanidine hydrochloride. Since the hydrophobic proteins are sequestered to the Det phase as long as the phase separation occurs, and if the hydrophobicity of the protein of interest is not affected by pH or denaturant, this technique can be also utilized to change buffers or to remove denaturants. When using enzymes or proteases which maintain activities in detergent solutions, TX114 can also be used to separate hydrophobic from the water-soluble hydrophilic moieties upon enzymatic digestion of proteins, as done by us using in vitro digestion of PrP with phosphatidylinositol-specific phospholipase C (Taguchi et al., 2013).

The purpose of this study was to evaluate the performance of standard-dose and low-dose cesium iodide (CsI)-doted amorphous silicon (a-Si) flat-panel detector technology (FDT) as compared with storage-phosphor technology (SPT) in the depiction of relevant anatomical structures in chest radiography. In 75 patients referred for thoracic CT, digital chest radiographs were randomly obtained with either SPT at a standard dose (speed class S400, n=25), standard-dose FDT (S400, n=25) or FDT at a low dose (S800, n=25). Five radiologists evaluated the visibility of eight pulmonary and mediastinal anatomical structures using a five-point rating scale. To determine statistically significant differences between the three groups, the Mann-Whitney U-test was employed. No statistically significant differences were found in the depiction of eight criteria between SPT and standard-dose or low-dose FDT chest radiographs. The performance of FDT S400 was equal to SPT for most criteria and better for retrocardiac structures and soft tissue. FDT S800 was inferior to both SPT and FDT S400. Standard-dose FDT is equivalent to SPT in the depiction of relevant anatomical structures of the chest. Our results also indicate that a dose reduction of 50% with FDT may result in small but not significant decrease of image quality.

To examine the role of stress-related 70-kDa heat shock proteins (Hsp-s) in Creutzfeldt–Jakob disease (CJD), we performed immunocytochemistry to detect Hsp-72 and Hsp-73, together with the abnormal (PrP Sc ) and the presumed cellular form (PrP C ) of the prion protein, and TUNEL method to measure cellular vulnerability in different brain regions in CJD and control cases. While Hsp-73 showed uniform distribution in all the examined samples, an increase in the number of Purkinje cells with prominent accumulation of Hsp-72 in the CJD group was observed. These neurons also showed intense PrP C staining, but TUNEL-positive nuclei were only detected in the granular (Hsp-72-negative) cell layer. Fewer cells of the inferior olivary nucleus were immunoreactive for Hsp-72 in CJD than in control cases, and regions showing severe spongiform change and gliosis exhibited fewer Hsp-72-immunoreactive neurons. Our results indicate that accumulation of the inducible Hsp-72 in certain cell types may be part of a cytoprotective mechanism, which includes preservation of proteins like PrP C .

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPcand the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPcand of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3.

Fragestellung: In dieser Ubersichtsarbeit soll das Konstruktionsprinzip und die Aufnahmebedingungen der Elektronenstrahl-Computertomographie (EBCT) dargestellt werden. Methodik: Im Gegensatz zum konventionellen CT werden beim EBCT die Schichtaufnahmen ohne mechanische Bauteile akquiriert. Dieses Konstruktionsprinzip erlaubt es, kurze Aufnahmezeiten zur Untersuchung eines anatomischen Bereichs (100 ms pro Schicht) oder bis zu 8 Schichthohen ohne Tischbewegung kurz nacheinander abzutasten (50 ms pro Schicht). Ergebnisse: Der Scanner kann je nach Einsatzgebiet im Einzelschicht-, kontinuierlichen Scan- und Multischicht-Modus betrieben werden. Der Einzelschicht-Modus wird zum Nachweis und zur Quantifizierung von Koronarkalzifikationen und fur die CT Angiographie der Koronargefase eingesetzt. Der kontinuierliche Scan-Modus entspricht dem Spiral-Modus bei dem konventionellen CT und kann bei Routinefragestellungen des Thorax und Abdomen eingesetzt werden. Der Multischichtbetrieb ist fur Funktionsuntersuchungen des Herzens ebenso wie fur Perfusionsuntersuchungen verschiedener Organe geeignet. Aufgrund der besonderen Scannergeometrie liegt die Strahlenbelastung bei einigen Betriebsarten jedoch deutlich uber der eines konventionellen Spiral-CT-Scanners. Schlusfolgerung: Kunftige Entwicklungen zielen auf dosiseffiziente Kollimation, hochauflosende Detektortechnologien und artefaktreduzierende Rekonstruktionsalgorithmen ab.

Summary Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS). The disease is increasingly important as a travel-related infection. Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures. In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays. The TOS N gene was amplified by reversetranscriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A. Sera with known TOS antibody status were used to evaluate the immunoblot assay. The expressed rTOS N protein was purified and used as antigen for immunobiots. By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified. No cross-reactivity was detected. The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis.

Ziel: Als virtuelle Koloskopie bezeichnet man eine neue Methode, die aus Bilddaten der radiologischen Schnittbildverfahren, CT, Elektronenstrahl-CT (EBT) und MRT mittels computerunterstutzter Bildnachverarbeitung simulierte dreidimensionale (3D-), endoskopieahnliche Bilder des Kolons erzeugt. Ziel unserer Untersuchung war die Bestimmung der Wertigkeit der virtuellen EBT-Koloskopie zum Nachweis von kolorektalen Polypen und Tumoren in vitro am Tumormodell und in Patientenstudien. Anhand publizierter Studien der virtuellen Koloskopie mit Spiral-CT und MRT sollen die verschiedenen Untersuchungstechniken dargestellt und verglichen werden. Material und Methode: 6 Polypen mit einem Durchmesser von 3–12 mm wurden nach Abheben und Umstechen der Mukosa in Schweinekolon gebildet. Nach Distension mit Raumluft wurde dieses Tumormodell mit EBT im „continuous volume scanning (CVS)-Mode” mit 3-mm-Kollimation und 3 verschiedenen Pitchverhaltnissen (0,4; 0,8; 1,5) gescannt; 20 Patienten mit positivem Hamocculttest wurden nach rektaler CO2-Insufflation und i. v.-Gabe von 1 mg Glucagon in Atemanhaltetechnik mit einem den ganzen Kolonrahmen abdeckenden CVS-Scan (13 s Untersuchungsdauer) untersucht. Endoskopische 3D-Durchflugsrekonstruktionen wurden von 2 unabhangigen Radiologen hinsichtlich des Vorliegens von Polypen oder Tumoren beurteilt. Die zu erwartende effektive Dosis fur die EBT-Koloskopie wurde uber Messungen mit einem Alderson-Phantom, welches mit Thermolumineszenzdosimetern bestuckt war, berechnet. Ergebnisse: Schon bei einem Pitch von 1,5 wurden alle Polypen im Tumormodell erkannt. Die Reduzierung des Pitch auf 0,8 oder 0,4 verbesserte nicht die Erkennbarkeit der Polypen auf den endoskopischen 3D-Rekonstruktionen. Alle Tumoren (n = 4) und Polypen (n = 3) der Patientenuntersuchungen wurden korrekt identifiziert. 2 falsch-positive Befunde wurden erhoben. Die effektive Dosis pro Scan errechnete sich zu maximal 3,2 mSv. Schlusfolgerungen: Unsere vorlaufigen Ergebnisse deuten darauf hin, das die virtuelle EBT-Koloskopie eine vielversprechende Methode zum Nachweis von kolorektalen Tumoren ist. Der Stellenwert der verschiedenen Techniken der virtuellen Koloskopie (Spiral-CT, EBT, MRT) mus erst noch bestimmt werden.

PURPOSE To compare softcopy and hardcopy reading of chest radiographs a software tool was designed for creating lesions with clearly defined size, location and contrast. METHOD An ROC study was performed using a set of chest radiographs with 300 simulated small nodules and linear details displayed on film and on a 1 K monitor. Six observers participated in the study. ROC curves were generated on the basis of 7200 decisions. RESULTS There was no significant difference in observer performance overall (AUC = Area under curve). Monitor: 0.84 +/- 0.08 vs. AUC, Film: 0.84 +/- 0.08). Slight, but not statistical significant differences were found in the detection of low-contrast lung nodules and of linear lung details. CONCLUSION Soft-copy reading using a high quality 1 K CRT display provides the same accuracy for the detection of subtle computer-simulated lesions as hardcopy reading. A software tool could be used for assigning optimal characteristics of CRT- and hardcopy displays using clearly defined lesions as a gold standard.

Variations in the major surface proteins (HBsAg) of hepatitis B virus (HBV) have been implicated in the high rate of reinfection in HBV-infected recipients of orthotopic liver transplantations (OLT). Sera from 6 OLT patients positive for HBsAg and from 3 recipients negative for it prior to transplantation were analyzed over several years, and 39 HBsAg sequences were compared. Despite anti-HBs immunoprophylaxis resulting in the disappearance of HBsAg, HBV DNA was detectable by a sensitive nested PCR in almost all sera. In 1 patient, a significant temporary shift in HBV subtypes was observed, indicating a mixed infection or the presence of multiple HBV populations in this patient; this was also true for other patients. Amino acid substitutions compared to wild-type HBV subtypes in 7 patients and variations within patients in 5 patients were detectable over time; the 'escape mutation' at amino acid position 145 was detected in 2 patients. Our data suggest that the high rate of reinfection in OLT recipients seems not to be associated with specific sequence variations in the major HBs gene, but shows a remarkable inter- and intraindividual variability. Obviously, no correlation between heterogeneity in this gene and clinical outcome was present. Copyright 1997 S. Karger AG, Basel

Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrP(c)) into the disease-associated isoform (PrP(Sc)) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids.

Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals.

Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrPSc of the host-encoded prion protein (PrPc). A profound conformational change of PrPc underlies formation of PrPSc and prion propagation involves conversion of PrPc substrate by direct interaction with PrPSc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrPC into PrPSc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrPSc recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated., Author Summary Prion diseases are deadly infectious diseases of the brain characterized by accumulation of a pathologic protein (PrPSc) which is derived from the normal prion protein (PrPc). Prions replicate by direct contact in a template-directed refolding process which involves conversion of PrPC into PrPSc. Identifying the modalities of this interaction can advance our molecular understanding of prion diseases. Like substrates and competitive inhibitors of enzymes, a conversion-incompetent PrP can inhibit conversion of normal PrPC, a phenomenon known as dominant-negative inhibition (DNI). Interestingly, some conversion-incompetent PrPs efficiently cause DNI but others do not, presumably depending on affinity for PrPSc and integrity of interaction interface. We utilized DNI to characterize the PrP-PrP interaction interface in cultured cells. We created a series of PrPs with internal deletions in the region between helix 1 and 2 and evaluated their DNI. We found an inverse correlation between deletion size and DNI which suggests that this region plays an important role in PrP-PrP interaction. We also found that such PrPs are subject to various cellular degradation pathways and that a fraction of them reaches the intracellular locale of prion conversion. Further investigation of such prion proteins might help elucidating the cellular mechanisms of the PrPC-PrPSc interaction.

Purpose Comparison of radiation doses in pulsed and continuous fluoroscopy to quantify the dose reduction by pulsed fluoroscopy. Further, the applicability of pulsed fluoroscopy in clinical routine has been evaluated. Materials and methods In a human pelvic phantom, the radiation dose (skin entry dose in cGycm2) was measured at two pulses per second (pps), 3 pps, 6 pps, 12 pps and continuous fluoroscopy mode, respectively, using image-intensifier entries of 38 cm, 25 cm, and 17 cm. 300 examinations were carried out, and the results of the different fluoroscopy modes were registered. Results Dose reduction depends on the image-intensifier entry. Compared to the radiation dose in continuous fluoroscopy, with 12 pps fluoroscopy the radiation dose can be reduced at a minimum of 51%, with 6 pps fluoroscopy to 40%, with 3 pps fluoroscopy to 20%, and with 2 pps fluoroscopy to a minimum of 14.5%. Clinical routine has shown that 78% of all examinations can be performed with 2 or 3 pps fluoroscopy mode. In 12.7% of the cases pulsed fluoroscopy of diverse frequencies was used, in an additional 2% combined with continuous fluoroscopy. Exclusively, continuous fluoroscopy has been employed in 2% of the cases. Conclusions Using pulsed fluoroscopy, an 80% reduction of the radiation dose compared to continuous fluoroscopy is possible. About 96% of all examinations can be performed with pulsed fluoroscopy of different pulse rate and without using continuous fluoroscopy.

Purpose To compare image quality of digital luminescence radiography with conventional film-screen techniques during excretion urography. Four field tests and ROC analysis for determining diagnostic value. Material and method 135 patients were included in a prospective study. Three independent observers judged the five minute (59 cases) or ten minute (76 cases) films after contrast injection using digital images as well as corresponding conventional images (five minutes-76 cases, ten minutes-59 cases). Results The digital technique provided better information concerning the renal parenchyma, the soft tissues and bone structures. Contrast enhanced detail was demonstrated equally well by both systems. The two systems had similar sensitivity but digital radiography showed higher specificity.

On comparing the individual imaging stages in conventional and digital image radiography it becomes evident that a significant advantage of the digital method is the possibility to adapt the image character and exposure dose individually to various problems requiring an answer. To take advantage of this it is imperative to optimise image processing with care, especially contrast processing. No systematic procedure based on objective criteria had existed to date. This paper describes a method of taking into account both the diagnostic significance of the structures to be imaged and their distribution within the relevant density range, in order to arrive at the best possible contrast conversion. A prerequisite is to know for every kind of imaging which distribution of grey values pertaining to the relevant structures can be expected before digital image processing (initial grey values). This distribution determines the most favourable contrast conversion. The processing parameters for several types of imaging were optimised by this method. The method is demonstrated via thoracic images of newborn and children.

Prion diseases are manifest as genetic, sporadic or infectious neurodegenerative disorders in humans and animals. The prolonged incubation times that accompany the transmission of prions between species are due, at least in part, to differences in prion protein (PrP) sequence. To examine the species barriers between non-human primates and humans, we sequenced the open reading frames (ORF) of 25 PrP genes from apes and monkeys. Comparison of the PrP genes of these animals with that of humans showed amino acid identities ranging from 92.9 to 99.6%. While phylograms of primate PrP sequences revealed a novel branching pattern for the apes, the genomic organization of all the primate PrP genes was similar, with the entire ORF contained within a single exon. Alignment of variant residues in primates, rodents and domestic animals showed no concordance with the mutations that segregate with human prion diseases or with polymorphisms that modulate disease in humans, mice and sheep. Most substitutions were conservative and, characteristically, clustered outside the four putative α-helical regions that are thought to form a four-helix bundle in the cellular isoform of PrP (PrP C ). Deletion of one of five Gly-Pro rich octarepeats from the N-terminus of PrP was seen in some species, while squirrel monkeys had an additional octarepeat; squirrel monkeys have been frequently used as experimental hosts for transmission of human prions. Alignment of primate and other mammalian PrP sequences suggests that codons between 90 and 130 have a profound influence on the transmissibility of prions from one species to another.

Here, we focus on the implications of two cellular degradation pathways on prion replication and clearance. The first one is autophagy which can have a promoting and inhibiting role in prion infection. Lysosomal prion clearance can be enhanced in vitro and in vivo by drug-induced activation of autophagy. More recent work revealed that a certain level of autophagy is needed for establishing acute and persistent prion infection, implicating that autophagy might represent a functional equivalent for a disaggregase function. Such one was postulated for seed fragmentation in prion propagation, similar to sonication in PMCA or Hsp104 in yeast prion biology. The second pathway described here is the proteasomal one. We have challenged various cell lines by inducing ER stress or compromising proteasomal activity and analyzed the effects on PrP metabolism strictly in the secretory pathway. Both events led to enhanced detection of PrP aggregates and significant increase of PrPSc in prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. These findings suggest a novel pathway which possibly provides additional substrate and template for prion formation when protein clearance by the proteasome is impaired and point to mechanisms which might play a role in prion de novo generation in sporadic prion diseases.

The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5 −/− mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease that mainly affects cattle. Transmission of BSE to humans caused a variant form of Creutzfeldt–Jakob disease. Following infection, the protease-resistant, disease-associated isoform of prion protein (PrPSc) accumulates in the central nervous system and in other tissues. Many countries have defined bovine tissues that may contain prions as specified risk materials, which must not enter the human or animal food chains and therefore must be discarded. Ultrasensitive techniques such as protein misfolding cyclic amplification (PMCA) have been developed to detect PrPSc when present in minuscule amounts that are not readily detected by other diagnostic methods such as immunohistochemistry or Western blotting. This study was conducted to determine when and where PrPSc can be found by PMCA in cattle orally challenged with BSE. A total of 48 different tissue samples from four cattle infected orally with BSE at various clinical stages of disease were examined using a standardized PMCA protocol. The protocol used brain homogenate from bovine PrP transgenic mice (Tgbov XV) as substrate and three consecutive rounds of PMCA. Using this protocol, PrPSc was found in the brain, spinal cord, nerve ganglia, optic nerve and Peyer’s patches. The presence of PrPSc was confirmed in adrenal glands, as well as in mesenteric lymph nodes – a finding that was reported recently by another group. Interestingly, additional positive results were obtained for the first time in the oesophagus, abomasum, rumen and rectum of clinically affected cattle.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field. © 2012 Landes Bioscience.

This article has been withdrawn (November 15, 2017), Background: In vivo folding could play an essential role in prion neurodegenerations. Results: Artificial mutants causing labile PrP folds when expressed in cells originate toxic CtmPrP featured by the absence of the intramolecular disulfide bond. Conclusion: Oxidative folding impairment facilitates the formation of the toxic PrP forms. Significance: Unveiling the mechanism facilitating the formation of toxic PrP forms is crucial for the understanding and prevention of prion disorders. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.