James Harper

Observation of metastable Aβ amyloid protofibrils by atomic force microscopy

Abstract Background : Brain amyloid plaque, a diagnostic feature of Alzheimers disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the β-amyloid protein (Aβ). As Aβ amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however. Results : Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Aβ variants Aβ1-40 and Aβ1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Aβ1-42 aggregates much more rapidly than Aβ1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Aβ amyloid protofibrils. Conclusions : Aβ protofibrils are likely to be intermediates in the in vitro assembly of Aβ amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Aβ aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Aβ protofibrils may be a useful therapeutic strategy.

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-β protein

BACKGROUND Amyloid plaques composed of the fibrillar form of the amyloid-beta protein (Abeta) are the defining neuropathological feature of Alzheimers disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Abeta to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Abeta amyloid protofibril, has been identified. Here, we report studies of Abeta amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy. RESULTS Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Abeta influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Abeta. Finally, branched and partially wound fibrils were observed. CONCLUSIONS The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.

Structural Aspects of Congo Red as an Inhibitor of Protease‐Resistant Prion Protein Formation

Abstract: Congo red (CR) has been shown to inhibit the accumulation in scrapie‐infected cells of prion protein (PrP) in the abnormal protease‐resistant form (PrP‐res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP‐res or its protease‐sensitive precursor (PrP‐sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP‐res formation in a simple cell‐free reaction composed predominantly of purified PrP‐res and PrP‐sen. Structurally modified CR analogues were also compared in both the cell‐free conversion reaction and scrapie‐infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2′ positions diminished the inhibitory potency by ≥10‐fold. In contrast, there was little effect of 3,3′ methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3′‐methyl‐CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl‐CR and amido‐CR. In contrast, 2,2′‐methyl‐CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP‐res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP‐res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP‐res formation by CR and related compounds.

Robust detection of individual forensic profiles in DNA mixtures

For a forensic identification method to be admissible in international courts, the probability of false match must be quantified. For comparison of individuals against complex mixtures using a panel of single nucleotide polymorphisms (SNPs), the probability of a random man not excluded, P(RMNE) is one admissible standard. While the P(RMNE) of SNP alleles has been previously studied, it remains to be rigorously defined and calculated for experimentally genotyped mixtures. In this report, exact P(RMNE) values were calculated for a range of complex mixtures, verified with Monte Carlo simulations, and compared alongside experimentally determined detection probabilities.

Sherlock's Toolkit: A forensic DNA analysis system

DNA sequence analysis has multiple forensic applications. The justice system currently uses sizes of STRs as well as mitochondrial DNA (mtDNA) for DNA evidence. With recent advancements in DNA sequencing technologies, inclusion of additional polymorphic loci, including SNPs, enable new useful analyses while maintaining backwards compatibility with STR sizing. Sherlocks Toolkit, developed by MIT Lincoln Laboratory, is an open source, scalable system for the integration and automation of STR and SNP-based analysis for high-throughput sequence data. The toolkit includes modules for a range of kinship, biogeographic ancestry, replicate, and mixture analyses.

Human CODIS STR loci profiling from HTS data

Human DNA identification is currently performed by amplifying a small, defined set of short tandem repeat (STR) loci (e.g. CODIS) and analyzing the size of the alleles present at those loci by capillary electrophoresis. High-throughput DNA sequencing (HTS) could enable the simultaneous analysis of many additional STR and single nucleotide polymorphism (SNP) loci, improving accuracy and discrimination. However, it is necessary to demonstrate that HTS can generate accurate data on the CODIS loci to enable backwards compatibility with the FBI NDIS database. Sequencing can also detect novel polymorphisms within alleles that migrate with identical sizes by capillary electrophoresis, improving allele discrimination, and enhancing human identification analysis. All CODIS alleles from an individual can be amplified in a single, multiplex PCR reaction, and combined with additional barcoded samples prior to sequencing. A computational tool for allele identification from multiplexed sequence data has been developed. With longer-read-length platforms, 99.6% allele calling accuracy can be achieved. In the course of STR sequencing protocol development, 12 novel allele sequences have been identified for multiple loci. Sequencing STR loci combined with SNPs will enable new forensic applications.