The Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-Cell Transmission Properties

Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)₁₅ DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)₁₅ with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-β sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)₁₅ DPR. Overall, our results show the structural and toxicity properties of GA DPR to facilitate future DPR-related therapeutic development.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis,molecular docking,acetylcholinesterase and butyrylcholinesterase inhibitory potential of thiazole analogs as new inhibitors for Alzheimer disease

A series of thirty (30) thiazole analogs were prepared, characterized by ¹H NMR, ¹³C NMR and EI-MS and evaluated for Acetylcholinesterase and butyrylcholinesterase inhibitory potential. All analogs exhibited varied butyrylcholinesterase inhibitory activity with IC₅₀ value ranging between 1.59 ± 0.01 and 389.25 ± 1.75 μM when compared with the standard eserine (IC₅₀, 0.85 ± 0.0001 μM). Analogs 15, 7, 12, 9, 14, 1, 30 with IC₅₀ values 1.59 ± 0.01, 1.77 ± 0.01, 6.21 ± 0.01, 7.56 ± 0.01, 8.46 ± 0.01, 14.81 ± 0.32 and 16.54 ± 0.21 μM respectively showed excellent inhibitory potential. Seven analogs 15, 20, 19, 24, 28, 30 and 25 exhibited good acetylcholinesterase inhibitory potential with IC50 values 21.3 ± 0.50, 35.3 ± 0.64, 36.6 ± 0.70, 44.81 ± 0.81, 46.36 ± 0.84, 48.2 ± 0.06 and 48.72 ± 0.91 μM respectively. All other analogs also exhibited well to moderate enzyme inhibition. The binding mode of these compounds was confirmed through molecular docking.     

The db mutation improves memory in younger mice in a model of Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Lepr^db/db) induces mixed or vascular dementia in mature to middle-aged APP^ΔNL/ΔNL x PS1^P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aβ content, which we have not observed at older ages. This was unlikely to be related to altered Aβ synthesis, as both β- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1β mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.     

Population density ofMonochamus alternatus adults (Coleoptera: Cerambycidae) and incidence of pine wilt disease caused byBursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Summary The seasonal occurrence ofMonochamus alternatus and newly weakened trees were investigated in aPinus thunbergii stand for 4 years. Adult beetles were present between June and September with a peak in their population occurring in early July followed by a decline then a period of about one month being in a steady number. The average number ofBursaphelenchus xylophilus (Nematoda), which is the causal agent of pine wilt disease, within beetles decreased as the season advanced. Pine trees newly weakened byB. xylophilus appeared between June and October, especially from August to October. The proportion of weakened or killed trees was directly proportional to the average beetle density per tree from June to August.     

Design and evaluation of control strategies for high cell density fermentations

A methodology for the design and evalution of bioprocess control strategies is presented. The strategies manage nutrient supply with demand and vary with the metabolic condition and phase of fermentation operation. Six carbon source addition strategies are based on different combinations of available measurements; they are described and evaluated under different operating conditions for yeast cultivation. It is concluded that a single control strategy is not the most appropriate under all possible operating conditions. An oxygen uptake rate-based control strategy performs better with a mean respiratory quotient (RQ) value less than 1.1 during an oxygen limitation than an ethanol control strategy which had a mean RQ of 14. The designed strategies and an approach of applying the strategy that best matches fermentation conditions consistently enables achievement of high cell densities 78.7 g DCW/L and yields 0.50 g DCW/g glucose as the mean values for three fermentations.     

On the parametrization of lateral dose profiles in proton radiation therapy

PurposeThe accurate evaluation of the lateral dose profile is an important issue in the field of proton radiation therapy. The beam spread, due to Multiple Coulomb Scattering (MCS), is described by the Molière's theory. To take into account also the contribution of nuclear interactions, modern Treatment Planning Systems (TPSs) generally approximate the dose profiles by a sum of Gaussian functions. In this paper we have compared different parametrizations for the lateral dose profile of protons in water for therapeutical energies: the goal is to improve the performances of the actual treatment planning.MethodsWe have simulated typical dose profiles at the CNAO (Centro Nazionale di Adroterapia Oncologica) beamline with the FLUKA code and validated them with data taken at CNAO considering different energies and depths. We then performed best fits of the lateral dose profiles for different functions using ROOT and MINUIT.ResultsThe accuracy of the best fits was analyzed by evaluating the reduced χ², the number of free parameters of the functions and the calculation time. The best results were obtained with the triple Gaussian and double Gaussian Lorentz–Cauchy functions which have 6 parameters, but good results were also obtained with the so called Gauss–Rutherford function which has only 4 parameters.ConclusionsThe comparison of the studied functions with accurate and validated Monte Carlo calculations and with experimental data from CNAO lead us to propose an original parametrization, the Gauss–Rutherford function, to describe the lateral dose profiles of proton beams.     

Phosphorylation of Bruton's tyrosine kinase by c-Abl

Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.     

Comparison of neurotoxicity of different aggregated forms of Aβ40, Aβ42 and Aβ43 in cell cultures

The abnormal deposition of amyloid‐β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.