H-1, N-15 and C-13 assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576

Psychological Health in Companies. An investigation into the relationships between work characteristics, job roles and psychological health in companies.

The research literature contains a significant amount of information on the psychological health of managers, however, relatively little has been written about the psychological health of workers. There has been a long held assumption that there is little difference in the psychological health of workers and managers. This study challenges that assumption and investigates differences in psychological health that appertain to job role and job characteristics determined by job role. This study tested two related hypotheses, firstly, that `managers have significantly better psychological health than workers¿; secondly, that `that better psychological health was largely due to the increased self esteem that comes from having a more socially desirable role¿. Initial analyses of the data indicated that hypothesis 1 was upheld, but when split by sex hypothesis 1 was only upheld for females and not males; similarly hypothesis 2 was only upheld for females. This result was attributed to the influence of two variables, the `attribution of blame¿ and `need for external validation¿, and the different moderator and mediator roles they play for males and females. The study contradicts the assertion that the psychological health of workers and managers is similar, if not the same, and uses the differences found to explain why research in this field has often produced conflicting results. Areas of potential future research are indicated

Insights Into Peptide Inhibition of Alpha-Synuclein Aggregation

α-Synuclein (aSyn) aggregation is an attractive target for therapeutic development fora range of neurodegenerative conditions, collectively termed synucleinopathies. Here,we probe the mechanism of action of a peptide 4554W, (KDGIVNGVKA), previously identified through intracellular library screening, to prevent aSyn aggregation and associated toxicity. We utilize NMR to probe association and identify that 4554Wassociates with a “partially aggregated” form of aSyn, with enhanced association occurring over time. We also report the ability of 4554W to undergo modification through deamidation of the central asparagine residue, occurring on the same timescale asaSyn aggregation in vitro,with peptide modification enhancing its association with aSyn.Additionally, we report that 4554W can act to reduce fibril formation of five Parkinson’s disease associated aSyn mutants. Inhibitory peptide binding to partially aggregated forms of aSyn, as identified here, is particularly attractive from a therapeutic perspective,as it would eliminate the need to administer the therapy at pre-aggregation stages,which are difficult to diagnose. Taken together the data suggest that 4554W could bea suitable candidate for future therapeutic development against wild-type, and most mutant aSyn aggregatio

NFT Certificates and Proof of Delivery for Fine Jewelry and Gemstones

Fine jewelry is a unique class of ornaments composed of precious metals and gemstones. Premium-grade metals such as gold, platinum, and sliver, and gemstones such as pearls, diamonds, rubies, and emeralds are used use to make fine jewelry. Paper-based certificates are typically issued by retailers and producers for fine jewelry and gemstones as a proof of origin, sale, ownership, history, and quality. However, paper certificates are subject to counterfeiting, loss, or theft. In this paper, we show how non-fungible tokens (NFTs) and Ethereum blockchain can be used for digital certification, proof of ownership, sale history, and quality, as well as proof of delivery for fine jewelry and gemstones.We present the proposed system design and architecture with sequence diagrams covering key interactions for jewelry production, purchase, and sale, along with algorithms related to NFT minting, auctioning, ownership management, and physical delivery. We demonstrate that our proposed NFT and blockchain-based solution can provide superior alternative in terms of verifiability, traceability, immutability, and security when compared with paper-based certification and traditional auctioning, delivery and ownership management. We make our developed smart contracts and testing scripts publicly available on GitHub

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis

Oxidative Stress Alters the Morphology and Toxicity of Aortic Medial Amyloid

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity

Comparisons with amyloid-β reveal an aspartate residue that stabilizes fibrils of the aortic amyloid peptide medin

Aortic medial amyloid (AMA) is the most common localized human amyloid, occurring in virtually all of the Caucasian population over the age of 50. The main protein component of AMA, medin, readily assembles into amyloid-like fibrils in vitro. Despite the prevalence of AMA, little is known about the self-assembly mechanism of medin or the molecular architecture of the fibrils. The amino acid sequence of medin is strikingly similar to the sequence of the Alzheimer's disease (AD) amyloid-beta (Aβ) polypeptides around the structural turn region of Aβ where mutations associated with familial, early onset AD, have been identified. D25 and K30 of medin align with residues D23 and K28 of Aβ that are known to form a stabilizing salt bridge in some fibril morphologies. Here we show that substituting D25 of medin with asparagine (D25N) impedes assembly into fibrils and stabilizes non-cytotoxic oligomers. Wild-type medin, by contrast, aggregates into β-sheet rich amyloid-like fibrils within 50 h. A structural analysis of wild-type fibrils by solid-state NMR suggests a molecular repeat unit comprising at least two extended β-strands, separated by a turn stabilized by a D25-K30 salt-bridge. We propose that D25 drives the assembly of medin by stabilizing the fibrillar conformation of the peptide, and is thus reminiscent of the influence of D23 on the aggregation of Aβ. Pharmacological comparisons of wild-type medin and D25N will help to ascertain the pathological significance of this poorly under-stood protein

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles