Conversion of Synthetic Aß to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.

Seeded strain-like transmission of ß-amyloid morphotypes in APP transgenic mice.

The polymorphic ß-amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral ß-amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop ß-amyloid depositions that differ in morphology, binding of amyloid conformation-sensitive dyes, and Aß40/Aß42 peptide ratio. To determine the nature of such ß-amyloid morphotypes, ß-amyloid-containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced ß-amyloid deposition with the morphological, conformational, and Aß40/Aß42 ratio characteristics of ß-amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced ß-amyloid deposits with the characteristics of ß-amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced ß-amyloid deposits, although less prominent, and the induced deposits were similar to the ß-amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aß in APP transgenic mice can be maintained by seeded conversion.

Highly potent soluble amyloid-ß seeds in human Alzheimer brain but not cerebrospinal fluid.

The soluble fraction of brain samples from patients with Alzheimer's disease contains highly biologically active amyloid-ß seeds. In this study, we sought to assess the potency of soluble amyloid-ß seeds derived from the brain and cerebrospinal fluid. Soluble Alzheimer's disease brain extracts were serially diluted and then injected into the hippocampus of young, APP transgenic mice. Eight months later, seeded amyloid-ß deposition was evident even when the hippocampus received subattomole amounts of brain-derived amyloid-ß. In contrast, cerebrospinal fluid from patients with Alzheimer's disease, which contained more than 10-fold higher levels of amyloid-ß peptide than the most concentrated soluble brain extracts, did not induce detectable seeding activity in vivo. Similarly, cerebrospinal fluid from aged APP-transgenic donor mice failed to induce cerebral amyloid-ß deposition. In comparison to the soluble brain fraction, cerebrospinal fluid largely lacked N-terminally truncated amyloid-ß species and exhibited smaller amyloid-ß-positive particles, features that may contribute to the lack of in vivo seeding by cerebrospinal fluid. Interestingly, the same cerebrospinal fluid showed at least some seeding activity in an in vitro assay. The present results indicate that the biological seeding activity of soluble amyloid-ß species is orders of magnitude greater in brain extracts than in the cerebrospinal fluid.

Aesthetic caries infiltration - Long-term masking efficacy after 6 years.

OBJECTIVES: This study aimed to evaluate the masking efficacy of caries infiltration technique of initial caries lesions (ICL) six years after debonding and single treatment. METHODS: In 10 adolescents, 74 ICL (ICDAS 2) in 74 teeth were treated by resin infiltration (Icon, DMG) at a mean (SD) of 1.2 (1.2) months after bracket removal. The etching procedure was performed up to 3 times. Standardized digital images were taken before treatment (T0), seven days (T7) and 6 years (T2190) after treatment. Outcomes included the evaluation of the color differences between carious and healthy enamel at T0, T7 and T2190 by quantitative colorimetric analysis (ΔE), ICDAS scores, quantitative light-induced fluorescence (QLF; ΔF,ΔQ,WS Area) and qualitative visual evaluation (5-point Likert-scale [deteriorated (1), unchanged (2), improved, but not satisfying (3), improved and no further treatment required (4), completely masked (5)). RESULTS: The median color difference ΔΕ0 (25th/75th percentiles) at T0 was 10.3 (8.56/13.0). At T7 a significant decrease was observed (ΔΕ7=3.7 (2.0/5.8); p<0.001; Friedmann-test; ICDAS p<0.001; Chi-square test). No significant changes based on ΔΕ (p=0.972; Friedmann-test) and ICDAS grading (p=0.511, chi-square test) were observed between T7 and T2190 (ΔΕ2190=2.9 (1.8/4.2)). Furthermore, at T2190 four experienced dentists classified 50% and 37% of the lesions as "improved and no further treatment required" and "completely masked", respectively (Fleiss kappa: T2190: 0.782 (substantial agreement)). CONCLUSION: Aesthetic caries infiltration can effectively mask post-orthodontic initial caries lesions for at least 6 years. These results for most of the teeth could not only be observed by quantitative but also by qualitative analysis. CLINICAL SIGNIFICANCE: Resin infiltration efficaciously masks post-orthodontic initial carious lesions. The optical improvement can be observed directly after treatment and remains stable for at least six years.

Soluble Aß seeds are potent inducers of cerebral ß-amyloid deposition.

Cerebral ß-amyloidosis and associated pathologies can be exogenously induced by the intracerebral injection of small amounts of pathogenic Aß-containing brain extract into young ß-amyloid precursor protein (APP) transgenic mice. The probable ß-amyloid-inducing factor in the brain extract has been identified as a species of aggregated Aß that is generated in its most effective conformation or composition in vivo. Here we report that Aß in the brain extract is more proteinase K (PK) resistant than is synthetic fibrillar Aß, and that this PK-resistant fraction of the brain extract retains the capacity to induce ß-amyloid deposition upon intracerebral injection in young, pre-depositing APP23 transgenic mice. After ultracentrifugation of the brain extract, <0.05% of the Aß remained in the supernatant fraction, and these soluble Aß species were largely PK sensitive. However, upon intracerebral injection, this soluble fraction accounted for up to 30% of the ß-amyloid induction observed with the unfractionated extract. Fragmentation of the Aß seeds by extended sonication increased the seeding capacity of the brain extract. In summary, these results suggest that multiple Aß assemblies, with various PK sensitivities, are capable of inducing ß-amyloid aggregation in vivo. The finding that small and soluble Aß seeds are potent inducers of cerebral ß-amyloidosis raises the possibility that such seeds may mediate the spread of ß-amyloidosis in the brain. If they can be identified in vivo, soluble Aß seeds in bodily fluids also could serve as early biomarkers for cerebral ß-amyloidogenesis and eventually Alzheimer's disease.

Induction of cerebral beta-amyloidosis: intracerebral versus systemic Abeta inoculation.

Despite the importance of the aberrant polymerization of Abeta in the early pathogenic cascade of Alzheimer's disease, little is known about the induction of Abeta aggregation in vivo. Here we show that induction of cerebral beta-amyloidosis can be achieved in many different brain areas of APP23 transgenic mice through the injection of dilute Abeta-containing brain extracts. Once the amyloidogenic process has been exogenously induced, the nature of the induced Abeta-deposition is determined by the brain region of the host. Because these observations are reminiscent of a prion-like mechanism, we then investigated whether cerebral beta-amyloidosis also can be induced by peripheral and systemic inoculations or by the intracerebral implantation of stainless steel wires previously coated with minute amounts of Abeta-containing brain extract. Results reveal that oral, intravenous, intraocular, and intranasal inoculations yielded no detectable induction of cerebral beta-amyloidosis in APP23 transgenic mice. In contrast, transmission of cerebral beta-amyloidosis through the Abeta-contaminated steel wires was demonstrated. Notably, plasma sterilization, but not boiling of the wires before implantation, prevented the induction of beta-amyloidosis. Our results suggest that minute amounts of Abeta-containing brain material in direct contact with the CNS can induce cerebral beta-amyloidosis, but that systemic cellular mechanisms of prion uptake and transport to the CNS may not apply to Abeta.

Next-generation biofuels: Survey of emerging technologies and sustainability issues.

Next-generation biofuels, such as cellulosic bioethanol, biomethane from waste, synthetic biofuels obtained via gasification of biomass, biohydrogen, and others, are currently at the center of the attention of technologists and policy makers in search of the more sustainable biofuel of tomorrow. To set realistic targets for future biofuel options, it is important to assess their sustainability according to technical, economical, and environmental measures. With this aim, the review presents a comprehensive overview of the chemistry basis and of the technology related aspects of next generation biofuel production, as well as it addresses related economic issues and environmental implications. Opportunities and limits are discussed in terms of technical applicability of existing and emerging technology options to bio-waste feedstock, and further development forecasts are made based on the existing social-economic and market situation, feedstock potentials, and other global aspects. As the latter ones are concerned, the emphasis is placed on the opportunities and challenges of developing countries in adoption of this new industry.