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Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

The amyloid-β (Aβ) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. (13)C, (15)N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.

This paper develops a statistics-based approach for analysis of transmission outage data. The method is applied to the Western Electricity Coordinating Council (WECC) transmission inventory and outage data collected and submitted by member utilities to the North American Electric Reliability Corporation's (NERC) Transmission Availability Data System (TADS). The developed model considers the random nature of Alternating Current (AC) circuit outages and introduces fourteen inventory attributes of the AC circuits as explanatory variables for the outage frequency. The main goal of the paper is to investigate statistical relationships between the number of automatic outages on AC circuits and AC circuit inventory attributes based on the TADS data from 2013–2018, which has proven to be sufficient in obtaining statistically valid results. Concepts associated with the statistical validation of performance indices are discussed with emphasis placed on analysis of distinct types of explanatory variables such as voltage class, mileage, number of terminals, circuits per structure, terrain, elevation, etc. [ABSTRACT FROM AUTHOR]

Background:There have been allegations in the courtroom that elevated serum lactic acid in trauma victims can yield a falsely elevated serum ethanol assay. Most hospitals utilize an indirect method of ethanol measurement where a serum sample is added to a mix of alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD+). This allows any ethanol in the patient’s serum to be metabolized to acetaldehyde, and in the process results in the reduction of NAD + to NADH. NADH is then measured using spectrophotometry. The courtroom allegation stems from the concept that oxidation of lactate to pyruvate by lactate dehydrogenase (LDH) results in the same molar-for-molar reduction of NAD + to NADH, and could therefore theoretically cause patients with elevated lactate and LDH to have a falsely elevated ethanol concentration. Methods:Patients with elevated lactic acid and LDH concentrations who presented to a university hospital from 20 April 2015 to 13 December 2015 were identified to provide possible test specimens. If a sufficient amount of serum was available, the sample was used to re-run the lactate and LDH concentration simultaneously with an enzymatic ethanol assay. Any samples that had elevated lactic acid and LDH concentrations on this retesting, and also yielded a positive ethanol concentration, were sent for confirmatory gas chromatography testing of ethanol concentrations. A control group of 20 samples with normal lactate and LDH were included. Results:A total of 37 samples were included in the final analysis. Only 4 patients had an elevated enzymatic ethanol concentration, and all 4 also had a measurable GC ethanol concentration. The lactate in this dataset ranged from 2.4 to 24.2 mmol/L, with a mean of 6.53 mmol/L (normal value 0.5–2.2). The LDH ranged from 242 to 8838 U/L with a mean of 1695 U/L (normal value 122–225 U/L). Twenty control samples were run on patients with normal lactate and LDH, none of which yielded a positive enzymatic ethanol result. Conclusions:This data does not support the contention that an elevated LDH and lactate can yield a false positive serum ethanol result as run by enzymatic ethanol assay in live patients presenting to the emergency department. [ABSTRACT FROM PUBLISHER]

This article examines the simple police cautioning disposal and argues that at present the theory and practice of the procedure do not coincide, resulting in unfairness, dissatisfaction and grounds for potential challenge. It is suggested that the process should be amended so that a police caution can be offered, with appropriate safeguards in place, prior to an admission being made. [ABSTRACT FROM AUTHOR]

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic 13 C enrichment of cholesterol with detection of protein-cholesterol 13 C- 13 C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A 13 C- 13 C double-quantum filter was employed to significantly simplify the 2D 13 C- 13 C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.

Purpose: Experimental tissue transplant studies reveal that lens development is directed by a series of early and late inductive interactions. These interactions impart a growing lens-forming bias within competent presumptive lens ectoderm that leads to specification and the commitment to lens fate. Relatively few genes are known which control these events. Identification of additional genes expressed during lens development may reveal key players in these processes and help to characterize these tissue properties., Methods: A large suite of genes has been isolated that are expressed during the process of cornea-lens transdifferentiation (lens regeneration) in Xenopus laevis. Many of these genes are also expressed during embryonic lens development. Genes were selected for expression analysis via in situ hybridization. This group consisted of clones with possible roles in cell determination and differentiation as well as novel clones without previous identities. The spatiotemporal expression of these genes in conjunction with previously described genes were correlated with key events during embryonic lens formation., Results: Eighteen of the thirty clones analyzed via in situ hybridization demonstrated observable expression in the developing lens. These genes were initially expressed in the presumptive lens ectoderm at a variety of timepoints throughout development. Expression is restricted to discrete time intervals during lens development. However, in most cases, expression was maintained throughout lens development after being initially upregulated., Conclusions: The expression of these genes suggests that a genetic hierarchy exists in which an increasing number of genes are upregulated and their expression is maintained throughout lens development. Suites of genes appear to be upregulated at specific timepoints during development, correlating with stages of lens induction, specification, commitment, lens placode formation, and lens differentiation, while suites at additional timepoints suggest that other, previously unreported stages exist as well. This analysis provides a genetic framework for characterizing these processes of lens development.