Disease-Associated Mutations in Tau Encode for Changes in Aggregate Structure Conformation.

The accumulation of tau fibrils is associated with neurodegenerative diseases, which are collectively termed tauopathies. Cryo-EM studies have shown that the packed fibril core of tau adopts distinct structures in different tauopathies, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. A subset of tauopathies are linked to missense mutations in the tau protein, but it is not clear whether these mutations impact the structure of tau fibrils. To answer this question, we developed a high-throughput protein purification platform and purified a panel of 37 tau variants using the full-length 0N4R splice isoform. Each of these variants was used to create fibrils in vitro, and their relative structures were studied using a high-throughput protease sensitivity platform. We find that a subset of the disease-associated mutations form fibrils that resemble wild-type tau, while others are strikingly different. The impact of mutations on tau structure was not clearly associated with either the location of the mutation or the relative kinetics of fibril assembly, suggesting that tau mutations alter the packed core structures through a complex molecular mechanism. Together, these studies show that single-point mutations can impact the assembly of tau into fibrils, providing insight into its association with pathology and disease.

Streamlined high-throughput cloning protocol to generate arrayed mutant libraries.

Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web app to automate the design of mutagenesis primers optimized for our cloning protocols in a 96-well plate format. The protocol allows most molecular biology labs to clone 96 mutants (from PCR to sequence ready plasmid) in 3 days.

Spectroscopic markers of neurodegeneration in the mesial prefrontal cortex predict survival in ALS.

Background and objective: N-acetylaspartate (NAA) and myo-inositol (mIns) are spectroscopic markers of neuronal integrity and astrogliosis, respectively. We performed a survival analysis to determine the prognostic value of the NAA/mIns metabolite ratio in ALS after a period of two and five years. Methods: Twenty-four patients with ALS (two with ALS-FTD) were recruited to participate in a high-field MR spectroscopy study of the mesial prefrontal cortex. Univariate and multivariate Cox proportional hazards analyses were used to assess NAA/mIns as a predictor of survival alongside other demographic and clinical measures. Census dates were set at two and five years after the time of MR scan for each patient. Survival curves were calculated using the Kaplan-Meier method. Results: After a five-year observation period, 19 patients had died and five were still alive. Median survival time from date of scan was 1.95 years. Univariate and multivariate Cox analysis showed NAA/mIns to be a significant independent predictor of survival at two years after scanning, but not at five years. Conclusion: Cerebral degeneration in the mesial prefrontal cortex as detected by the NAA/mIns metabolite ratio is predictive of survival in ALS in a time-dependent manner.

Accessing thermoplastic processing windows in metallic glasses using rapid capacitive discharge.

The ability of the rapid-capacitive discharge approach to access optimal viscosity ranges in metallic glasses for thermoplastic processing is explored. Using high-speed thermal imaging, the heating uniformity and stability against crystallization of Zr35Ti30Cu7.5Be27.5 metallic glass heated deeply into the supercooled region is investigated. The method enables homogeneous volumetric heating of bulk samples throughout the entire supercooled liquid region at high rates (~10(5) K/s) sufficient to bypass crystallization throughout. The crystallization onsets at temperatures in the vicinity of the "crystallization nose" were identified and a Time-Temperature-Transformation diagram is constructed, revealing a "critical heating rate" for the metallic glass of ~1000 K/s. Thermoplastic process windows in the optimal viscosity range of 10(0)-10(4) Pa · s are identified, being confined between the glass relaxation and the eutectic crystallization transition. Within this process window, near-net forging of a fine precision metallic glass part is demonstrated.