'An Apple a Day'?: Psychiatrists, Psychologists and Psychotherapists Report Poor Literacy for Nutritional Medicine: International Survey Spanning 52 Countries.

Nutritional interventions have beneficial effects on certain psychiatric disorder symptomatology and common physical health comorbidities. However, studies evaluating nutritional literacy in mental health professionals (MHP) are scarce. This study aimed to assess the across 52 countries. Surveys were distributed via colleagues and professional societies. Data were collected regarding self-reported general nutrition knowledge, nutrition education, learning opportunities, and the tendency to recommend food supplements or prescribe specific diets in clinical practice. In total, 1056 subjects participated in the study: 354 psychiatrists, 511 psychologists, 44 psychotherapists, and 147 MHPs in-training. All participants believed the diet quality of individuals with mental disorders was poorer compared to the general population (p < 0.001). The majority of the psychiatrists (74.2%) and psychologists (66.3%) reported having no training in nutrition. Nevertheless, many of them used nutrition approaches, with 58.6% recommending supplements and 43.8% recommending specific diet strategies to their patients. Only 0.8% of participants rated their education regarding nutrition as 'very good.' Almost all (92.9%) stated they would like to expand their knowledge regarding 'Nutritional Psychiatry.' There is an urgent need to integrate nutrition education into MHP training, ideally in collaboration with nutrition experts to achieve best practice care.

Fracture resistance of endodontically treated teeth restored with resin post reinforced with glass fiber.

This study aims to determine whether the design of resin posts reinforced with glass fiber (FRC) and Reporfost (Angelus, Londrina, PR, Brazil) significantly improves the fracture resistance of endodontically treated teeth restored through this method.A batch of 30 maxillary monoradicular teeth (15 central incisors, 15 canines) were treated endodontically by step-back technique (apical enlargement 40-K file) sealed with Sealapex (Kerr Corporation, Orange, US) and gutta-percha by lateral condensation, cold. They were divided into two equal groups, prepared for cementing the FRC posts. The Exacto posts (Angelus, Londrina, PR, Brazil) in group 1 and the Reforpost posts (Angelus; Londrina; PR, Brazil) were cemented with dual cure resin cement Breeze Self-Adhesive Resin Cement (Pentron Clinical, Orange, US). Fracture resistance testing was performed on the crown-apical axial direction, using the Hounsfield / Tinius Olsen H1-KS, PA, USA mechanical testing apparatus. The behavior of each tooth-post assembly was recorded as a graph. The statistical analysis was done using one way ANOVA (α=0.05). The differences between the Exacto post group and the Reforpost post group are not statistically significant (p = 0.466). The maximum force recorded was 970 N and the minimum 186N. The mean force at which the fracture occurred was approximately 500N for both groups. The strain test showed that modifying the Reforpost post design did not improve the fracture resistance parameters of the tooth-post assembly through increasing the surface friction or maintaining adhesion to the walls of the root dentin.

Modulation of Creutzfeldt-Jakob disease prion propagation by the A224V mutation.

OBJECTIVE: Mutations in the gene encoding the prion protein (PrP) are responsible for approximately 10 to 15% of cases of prion disease in humans, including Creutzfeldt-Jakob disease (CJD). Here, we report on the discovery of a previously unreported C-terminal PrP mutation (A224V) in a CJD patient exhibiting a disease similar to the rare VV1 subtype of sporadic (s) CJD and investigate the role of this mutation in prion replication and transmission. METHODS: We generated transgenic (Tg) mice expressing human PrP with the V129 polymorphism and A224V mutation, denoted Tg(HuPrP,V129,A224V) mice, and inoculated them with different subtypes of sCJD prions. RESULTS: Transmission of sCJD VV2 or MV2 prions was accelerated in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice, with incubation periods of ∼110 and ∼210 days, respectively. In contrast, sCJD MM1 prions resulted in longer incubation periods in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice (∼320 vs. ∼210 days). Prion strain fidelity was maintained in Tg(HuPrP,V129,A224V) mice inoculated with sCJD VV2 or MM1 prions, despite the altered replication kinetics. INTERPRETATION: Our results suggest that A224V is a risk factor for prion disease and modulates the transmission behavior of CJD prions in a strain-specific manner, arguing that residues near the C-terminus of PrP are important for controlling the kinetics of prion replication.

Conserved properties of human and bovine prion strains on transmission to guinea pigs.

The first transmissions of human prion diseases to rodents used guinea pigs (Gps, Cavia porcellus). Later, transgenic mice expressing human or chimeric human/mouse PrP replaced Gps, but the small size of the mouse limits some investigations. To investigate the fidelity of strain-specific prion transmission to Gps, we inoculated 'type 1' and 'type 2' prion strains into Gps, and we measured the incubation times and determined the strain-specified size of the unglycosylated, protease-resistant (r) PrP(Sc) fragment. Prions passaged once in Gps from cases of sporadic (s) Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L mutation were used, as well as human prions from a variant (v) CJD case, bovine prions from bovine spongiform encephalopathy (BSE) and mouse-passaged scrapie prions. Variant CJD and BSE prions transmitted to all the inoculated Gps with incubation times of 367 ± 4 and 436 ± 28 days, respectively. On second passage in Gps, vCJD and BSE prions caused disease in 287 ± 4 and 310 ± 4 days, whereas sCJD and GSS prions transmitted in 237 ± 4 and 279 ± 19 days, respectively. Although hamster Sc237 prions transmitted to two of three Gps after 574 and 792 days, mouse-passaged RML and 301V prion strains, the latter derived from BSE prions, failed to transmit disease to Gps. Those Gps inoculated with vCJD or BSE prions exhibited 'type 2' unglycosylated, rPrP(Sc) (19 kDa), whereas those receiving sCJD or GSS prions displayed 'type 1' prions (21 kDa), as determined by western blotting. Such strain-specific properties were maintained in Gps as well as mice expressing a chimeric human/mouse transgene. Gps may prove particularly useful in further studies of novel human prions such as those causing vCJD.

Generation of antisera to purified prions in lipid rafts.

Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP(C) into alternately folded PrP(Sc). Immunoassays toward pre-clinical detection of infectious PrP(Sc) have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP(Sc) selective antibodies. We report a method to purify infectious PrP(Sc) from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP(Sc) was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM <20-fold and purified DRM immunogen <40-fold) relative to 1% crude brain homogenate. Purification of PrP(Sc) from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP(Sc) antigen was performed using wild-type (wt) and Prnp(0/0) mice, both on Balb/cJ background. A robust immune response against PrP(Sc) was observed in all inoculated Prnp(0/0) mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP(C) and PrP(Sc), while binding to other brain-derived protein was not observed. In contrast, the PrP(Sc) inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.

Conformation-dependent high-affinity monoclonal antibodies to prion proteins.

Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP(Sc), an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP(Sc) commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP(Sc) from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K(D) values for mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.

Prion proteins with pathogenic and protective mutations show similar structure and dynamics.

Conformational change in the prion protein (PrP) is thought to be responsible for a group of rare but fatal neurodegenerative diseases of humans and other animals, including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. However, little is known about the mechanism by which normal cellular PrPs initiate and propagate the conformational change. Here, we studied backbone dynamics of the inherited pathogenic mutants (P101L and H186R), protective mutants (Q167R and Q218K), and wild-type mouse PrP(89-230) at pH 5.5 and 3.5. Mutations result in minor chemical shift changes around the mutation sites except that H186R induces large chemical shift changes at distal regions. At lower pH values, the C-terminal half of the second helix is significantly disordered for the wild-type and all mutant proteins, while other parts of the protein are essentially unaffected. This destabilization is accompanied by protonation of the partially exposed histidine H186 in the second helix of the wild-type protein. This region in the mutant protein H186R is disordered even at pH 5.5. The wild-type and mutant proteins have similar microsecond conformational exchange near the two beta-strands and have similar nanosecond internal motions in several regions including the C-terminal half of the second helix, but only wild type and P101L have extensive nanosecond internal motions throughout the helices. These motions mostly disappear at lower pH. Our findings raise the possibility that the pathogenic or dominant negative mutations exert their effects on some non-native intermediate form such as PrP* after conversion of cellular PrP (PrP(C)) into the pathogenic isoform PrP(Sc) has been initiated; additionally, formation of PrP(Sc) might begin within the C-terminal folded region rather than in the disordered N-terminal region.

Cryo-immunogold electron microscopy for prions: toward identification of a conversion site.

Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).

Human prions and plasma lipoproteins.

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). The polyoxometalate phosphotungstic acid has been used to separate PrP(Sc) from its precursor PrP(C) by selective precipitation; notably, native PrP(Sc) has not been solubilized by using nondenaturing detergents. Because of the similarities between PrP(Sc) and lipoproteins with respect to hydrophobicity and formation of phosphotungstic acid complexes, we asked whether these molecules are bound to each other in blood. Here we report that prions from the brains of patients with sporadic Creutzfeldt-Jakob disease (CJD) bind to very low-density (VLDL) and low-density (LDL) lipoproteins but not to high-density lipoproteins (HDL) or other plasma components, as demonstrated both by affinity assay and electron microscopy. Immunoassays demonstrated that apolipoprotein B (apoB), which is the major protein component of VLDL and LDL, bound PrP(Sc) through a highly cooperative process. Approximately 50% of the PrP(Sc) bound to LDL particles was released after exposure to 4 M guanidine hydrochloride at 80 degrees C for 20 min. The apparent binding constants of native human (Hu) PrP(Sc) or denatured recombinant HuPrP(90-231) for apoB and LDL ranged from 28 to 212 pM. Whether detection of PrP(Sc) in VLDL and LDL particles can be adapted into an antemortem diagnostic test for prions in the blood of humans, livestock, and free-ranging cervids remains to be determined.

Vascular tumors in the orbit--capillary and cavernous hemangiomas.

Vascular tumors in the orbit result from new formation of vessels, proliferation of tissue components of the vessel wall, and hyperplasia of cellular elements ordinarily concerned with the genesis of vascular tissue. These vasculogenic lesions constitute the largest group of primary orbital tumors; we present the capillary hemangioma and the cavernous hemangioma.

Search for a prion-specific nucleic acid.

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.

Diagnosis of human prion disease.

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrPSc) used limited proteolysis to digest the precursor cellular PrP (PrPC). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrPSc in all 10-24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, approximately 90% of the total PrPSc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrPC. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

Immunoglobulins in urine of hamsters with scrapie.

In the prion diseases, a prolonged, asymptomatic incubation period precedes the onset of neurologic dysfunction. At present, a noninvasive test is not available for the presymptomatic diagnosis of prion disease, and thus the report of a test for prions using urine has been of great interest (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482). Using Western immunoblots with the anti-prion protein (PrP) 3F4 monoclonal antibody and an anti-mouse IgG secondary antibody, a protease-resistant PrP was reported in the urine of Syrian hamsters and humans with prion disease. Here we have demonstrated that this purportedly "protease-resistant PrP" band in the urine of diseased hamsters is detectable using the anti-mouse IgG secondary antibody in the absence of the 3F4 monoclonal antibody. Mass spectrometric analysis identified an immunoglobulin light chain in the band but found no PrP peptides. No similar band was found in the urine of uninfected hamsters or in brain homogenates from normal or prion-infected hamsters. Moreover, the band in the urine of infected hamsters was not detected using two chimeric human-mouse recombinant anti-PrP antibody fragments followed by an anti-human IgG secondary antibody. Our results indicate that the band detected under previously published conditions is due to the cross-reactivity of the anti-mouse IgG antibody with IgG light chains and possibly heavy chain fragments in urine, but not with PrP.

Conformation of PrP(C) on the cell surface as probed by antibodies.

We have investigated the conformation of Syrian hamster PrP(C) on the surface of transfected CHO cells by performing cross-competition experiments between a set of nine monoclonal antibody fragments (Fab) directed to defined epitopes throughout the protein. No competition was observed between antibodies recognizing epitopes located within the unstructured N-terminal portion of PrP(C) and those recognizing epitopes located within the ordered C-terminal half of the molecule. However, competition was observed between antibodies recognizing overlapping epitopes and between antibodies recognizing epitopes lying adjacent to one another in the PrP sequence. Titrating the reactivity of each Fab against cell-surface PrP(C) revealed a clear heterogeneity in the accessibility of different specific epitopes. Fab D18, recognizing sequence incorporating the first alpha-helix of PrP(C), bound the largest fraction of the cell-surface PrP population. In contrast, Fab E123, binding an epitope at the extreme N terminus of PrP, and Fab 13A5, binding an epitope in the central region of PrP, were able to recognize fewer than half the number of PrP(C) molecules bound by Fab D18. The pattern of antibody reactivity we observed may, in part, result from N-terminal truncation of a proportion of PrP(C) molecules found at the cell surface. However, truncation cannot account for the marked disparity between exposure of the Fab D18 and 13A5 epitopes, which lie adjacent in the PrP sequence. The relative inaccessibility of the 13A5 epitope likely reflects either PrP(C)-PrP(C) interaction, interaction between PrP(C) and other constituents on the cell membrane, or the existence of PrP(C) subspecies with distinct conformations.

Structural studies of the scrapie prion protein by electron crystallography.

Because the insolubility of the scrapie prion protein (PrP(Sc)) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 A resolution. By comparing projection maps of PrP 27-30 and PrP(Sc)106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP(Sc). Only models featuring parallel beta-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.