Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the Aβ fly model and the AβPP-BACE1 fly model. In the Aβ fly model, the Aβ peptide is fused to a secretion sequence and directly overexpressed. In the AβPP-BACE1 model, human AβPP and human BACE1 are expressed in the fly, resulting in in vivo production of Aβ peptides and other AβPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the Aβ fly model, this correlates with high Aβ1-42 levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the AβPP-BACE1 fly model, neuronal cell death correlates with low Aβ1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of AβPP/Aβ antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the AβPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments

Probing the unfolded protein response in long-lived naked mole-rats.

The long-living naked mole-rat (NMR) shows negligible senescence and resistance to age-associated diseases. Recent evidence, based on protein-level assays, suggests that enhanced protein homeostasis machinery contributes to NMR stress-resistance and longevity. Here, we develop NMR-specific, transcriptional assays for measuring the unfolded protein response (UPR), a component of ER proteostasis. By varying doses and response times of pharmacological ER stressors applied to NMR kidney fibroblasts, we probe the NMR UPR in detail, demonstrating that NMR fibroblasts have a higher UPR activation threshold compared to mouse fibroblasts under mild ER-stress induction; whereas temporal analysis reveals that severe ER-stress induction results in no comparative differences. Probing NMR UPR activation with our robust assays may lead to insights into the proteostasis and ageing relationship.Herchel Smith PhD Research Scholarship Rosetrees Trust Gates Cambridge Trust scholarshi

Analysis of the native structure, stability and aggregation of biotinylated human lysozyme.

Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions

Engineering mono- and multi-valent inhibitors on a modular scaffold.

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.BBSRC Doctoral Training Programme (DTP) scholarship Oliver Gatty Studentship AstraZeneca PhD studentship. UK Medical Research Foundation. CRUK Pioneer Award (C17838/A22676) CRUK BTERP Award (C17838/A27225) Leverhulme Trust (RPG-2014-089) Cambridge Newton Trust BBSRC project grant (BB/T002697/1)

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol

Measurements of time-dependent CP asymmetries in B→D∗∓π±B \to D^{*\mp} \pi^{\pm} decays using a partial reconstruction technique

We report results on time-dependent CP asymmetries in $B \to D^{*\mp}\pi^{\pm}$ decays based on a data sample containing 657 {\times} $10^6$ $B\bar{B}$ pairs collected with the Belle detector at the KEKB asymmetric-energy $e^+ e^-$ collider at the $\Upsilon(4S)$ resonance. We use a partial reconstruction technique, wherein signal $B \to D^{*\mp}\pi^{\pm}$ events are identified using information only from the fast pion from the B decay and the slow pion from the subsequent decay of the $D^{*\mp}$, where the former (latter) corresponds to $D^{*+} (D^{*-})$ final states. We obtain CP violation parameters $S^+ = +0.061 \pm 0.018(\mathrm{stat}) \pm 0.012(\mathrm{syst})$ and $S^- = +0.031 \pm 0.019(\mathrm{stat}) \pm 0.015(\mathrm{syst})$.Comment: 8 pages, 5 figures, 2 tables, submitted to Physical Review D (RC

Measurement of the decay B0→π−ℓ+νB^0\to\pi^-\ell^+\nu and determination of ∣Vub∣|V_{ub}|

We present a measurement of the charmless semileptonic decay $B^0\to\pi^-\ell^+\nu$ using a data sample containing 657$\times 10^6$ $B\bar{B}$ events collected with the Belle detector at the KEKB asymmetric-energy $e^+e^-$ collider operating near the $\Upsilon(4S)$ resonance. We determine the total branching fraction of the decay, $\mathcal{B}(B^0\to\pi^-\ell^+\nu)=(1.49\pm 0.04{(\mathrm{stat})}\pm 0.07{(\mathrm{syst})})\times 10^{-4}$. We also report a new precise measurement of the differential decay rate, and extract the Cabibbo-Kobayashi-Maskawa matrix element $|V_{ub}|$ using model-independent and -dependent approaches. From a simultaneous fit to the measured differential decay rate and lattice QCD results, we obtain $|V_{ub}|=(3.43\pm 0.33)\times 10^{-3}$, where the error includes both statistical and systematic uncertainties.Comment: 8 pages, 3 figures, Submitted to PRD(RC

Search for CP violation in the decays D(s)+→KS0π+D^+_{(s)} \to K_S^0\pi^+ and D(s)+→KS0K+D^+_{(s)} \to K_S^0K^+

We have searched for CP violation in the charmed meson decays $D^{+}_{(s)}\to K^0_S\pi^+$ and $D^{+}_{(s)}\to K^0_S K^+$ using 673 fb$^{-1}$ of data collected with the Belle detector at the KEKB asymmetric-energy $e^+e^-$ collider. No evidence for CP violation is observed. We report the most sensitive CP asymmetry measurements to date for these decays: $A_{CP}^{D^+\to K^0_S\pi^+}=(-0.71\pm0.19\pm0.20)$, $A_{CP}^{D^+_s\to K^0_S\pi^+}=(+5.45\pm2.50\pm0.33)$, $A_{CP}^{D^+\to K^0_S K^+}=(-0.16\pm0.58\pm0.25)$, and $A_{CP}^{D^+_s\to K^0_S K^+}=(+0.12\pm0.36\pm0.22)$, where the first uncertainties are statistical and the second are systematic

Search for CP Violation in D Meson Decays to phi pi+

We search for CP violation in Cabibbo-suppressed charged D meson decays by measuring the difference between the CP violating asymmetries for the Cabibbo-suppressed decays D+ -> K+K-pi+ and the Cabibbo-favored decays Ds -> K+K-pi+ in the K+K- mass region of the phi resonance. Using 955/fb of data collected with the Belle detector we obtain A_CP(D+ -> phi pi+) = (+0.51 +- 0.28 +- 0.05)%. The measurement improves the sensitivity of previous searches by more than a factor of five. We find no evidence for direct CP violation.Comment: submitted to PR

Observation of B+ -> Dbar*0 tau+ nu_tau and Evidence for B+ -> Dbar^0 tau+ nu_tau at Belle

We present measurements of B+ -> Dbar*0 tau+ nu_tau and B+ -> Dbar^0 tau+ nu_tau decays in a data sample of 657 x 10^6 BBbar pairs collected with the Belle detector at the KEKB asymmetric-energy e+e- collider. We find 446^{+58}_{-56} events of the decay B+ -> Dbar*0 tau+ nu_tau with a significance of 8.1 standard deviations, and 146^{+42}_{-41} events of the decay B+ -> Dbar0 tau+ nu_tau with a significance of 3.5 standard deviations. The latter signal provides the first evidence for this decay mode. The measured branching fractions are B(B+ -> Dbar*0 tau+ nu_tau)=(2.12^{+0.28}_{-0.27} (stat) +- 0.29 (syst)) % and B(B+ -> Dbar0 tau+ nu_tau)=(0.77 +- 0.22 (stat) +- 0.12 (syst)) %.Comment: 6 pages, 4 figures, submitted to Phys. Rev. Let