MRI sensing based on the displacement of paramagnetic ions from chelated complexes

We introduce a mechanism for ion sensing by MRI in which analytes compete with paramagnetic ions for binding to polydentate chelating agents. Displacement of the paramagnetic ions results in alteration of solvent interaction parameters and consequent changes in relaxivity and MRI contrast. The MRI changes can be tuned by the choice of chelator. As an example, we show that calcium-dependent displacement of Mn[superscript 2+] ions bound to EGTA and BAPTA results in a T[subscript 1]-weighted MRI signal increase, whereas displacement from calmodulin results in a signal decrease. The changes are ion selective and can be explained using relaxivity theory. The ratio of T[subscript 2] to T[subscript 1] relaxivity is also calcium-dependent, indicating the feasibility of “ratiometric” analyte detection, independent of the probe concentration. Measurement of paramagnetic ion displacement effects could be used to determine analyte ion concentrations with spatial resolution in opaque specimens.National Institutes of Health (U.S.) (grant DP2-OD2441)National Institutes of Health (U.S.) (grant R01-GM65519)McGovern Institute for Brain Research at MIT. Neurotechnology (MINT) Progra

Mapping of functionalized regions on carbon nanotubes by scanning tunneling microscopy

Scanning tunneling microscopy (STM) gives us the opportunity to map the surface of functionalized carbon nanotubes in an energy resolved manner and with atomic precision. But this potential is largely untapped, mainly due to sample stability issues which inhibit reliable measurements. Here we present a simple and straightforward solution that makes away with this difficulty, by incorporating the functionalized multiwalled carbon nanotubes (MWCNT) into a few layer graphene - nanotube composite. This enabled us to measure energy resolved tunneling conductance maps on the nanotubes, which shed light on the level of doping, charge transfer between tube and functional groups and the dependence of defect creation or functionalization on crystallographic orientation.Comment: Keywords: functionalization, carbon nanotubes, few layer graphene, STM, CITS, ST

An ancestral 10-bp repeat expansion in VWA1 causes recessive hereditary motor neuropathy

The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6–83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses

Role of water in Protein Aggregation and Amyloid Polymorphism

A variety of neurodegenerative diseases are associated with the formation of amyloid plaques. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Most experimental and simulation studies have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked. In this Account, we provide a perspective on how interactions with water affect folding landscapes of A$\beta$ monomers, A$\beta_{16-22}$ oligomer formation, and protofilament formation in a Sup35 peptide. Simulations show that the formation of aggregation-prone structures (N$^*$) similar to the structure in the fibril requires overcoming high desolvation barrier. The mechanism of protofilament formation in a polar Sup35 peptide fragment illustrates that water dramatically slows down self-assembly. Release of water trapped in the pores as water wires creates protofilament with a dry interface. Similarly, one of the main driving force for addition of a solvated monomer to a preformed fibril is the entropy gain of released water. We conclude by postulating that two-step model for protein crystallization must also hold for higher order amyloid structure formation starting from N$^*$. Multiple N$^*$ structures with varying water content results in a number of distinct water-laden polymorphic structures. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow down fibril growth rates in hydrophilic sequences.Comment: 27 pages, 4 figures; Accounts of Chemical Research, 201

Atypical parkinsonism-associated retromer mutant alters endosomal sorting of specific cargo proteins

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand–containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism