Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ‐secretase

γ‐Secretase plays a central role in the generation of the Alzheimer disease‐causing amyloid β‐peptide (Aβ) from the β‐amyloid precursor protein (APP) and is thus a major Alzheimer′s disease drug target. As several other γ‐secretase substrates including Notch1 and CD44 have crucial signaling functions, an understanding of the mechanism of substrate recognition and cleavage is key for the development of APP selective γ‐secretase‐targeting drugs. The γ‐secretase active site domain in its catalytic subunit presenilin (PS) 1 has been implicated in substrate recognition/docking and cleavage. Highly critical in this process is its GxGD active site motif, whose invariant glycine residues cannot be replaced without causing severe functional losses in substrate selection and/or cleavage efficiency. Here, we have investigated the contribution of the less well characterized residue x of the motif (L383 in PS1) to this function. Extensive mutational analysis showed that processing of APP was overall well‐tolerated over a wide range of hydrophobic and hydrophilic mutations. Interestingly, however, most L383 mutants gave rise to reduced levels of Aβ_37–39 species, and several increased the pathogenic Aβ_42/43 species. Several of the Aβ_42/43‐increasing mutants severely impaired the cleavages of Notch1 and CD44 substrates, which were not affected by any other L383 mutation. Our data thus establish an important, but compared with the glycine residues of the motif, overall less critical functional role for L383. We suggest that L383 and the flanking glycine residues form a spatial arrangement in PS1 that is critical for docking and/or cleavage of different γ‐secretase substrates.     

Design,synthesis and biological evaluation of novel aminothiazoles as antiviral compounds acting against human rhinovirus

We describe here the design, synthesis and biological evaluation of antiviral compounds acting against human rhinovirus (HRV). A series of aminothiazoles demonstrated pan-activity against the HRV genotypes screened and productive structure–activity relationships. A comprehensive investigational library was designed and performed allowing the identification of potent compounds with lower molecular weight and improved ADME profile. 31d-1, 31d-2, 31f showed good exposures in CD-1 mice. The mechanism of action was discovered to be a host target: the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). The identification of the pan-HRV active compound 31f combined with a structurally distinct literature compound T-00127-HEV1 allowed the assessment of target related tolerability of inhibiting this kinase for a short period of time in order to prevent HRV replication.     

The genetic rescue of two bottlenecked South Island robin populations using translocations of inbred donors

Populations forced through bottlenecks typically lose genetic variation and exhibit inbreeding depression. ‘Genetic rescue’ techniques that introduce individuals from outbred populations can be highly effective in reversing the deleterious effects of inbreeding, but have limited application for the majority of endangered species, which survive only in a few bottlenecked populations. We tested the effectiveness of using highly inbred populations as donors to rescue two isolated and bottlenecked populations of the South Island robin (Petroica australis). Reciprocal translocations significantly increased heterozygosity and allelic diversity. Increased genetic diversity was accompanied by increased juvenile survival and recruitment, sperm quality, and immunocompetence of hybrid individuals (crosses between the two populations) compared with inbred control individuals (crosses within each population). Our results confirm that the implementation of ‘genetic rescue’ using bottlenecked populations as donors provides a way of preserving endangered species and restoring their viability when outbred donor populations no longer exist.     

Growth performance,carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets

A growth trial with 38 weaners (castrated male pigs) was designed to compare the growth performance and carcass quality of pigs fed diets containing either soy bean meal or soy protein concentrate in a pair-feeding design. Soy bean meal (SBM) and soy protein concentrate (SPC) differed in isoflavone (daidzein plus genistein) content (782?μg/g in SBM and 125?μg/g in SPC, respectively). During the experiment, all pigs were fed four-phases-diets characterized by decreasing protein concentrations with increasing age (weaner I, weaner II, grower, finisher diets). Rations of control and experimental groups were isoenergetic, isonitrogenous, and isoaminogen. The weanling pigs with an initial live weight of 8.4?±?1.1?kg were allotted to flat deck boxes. During the growing/finishing period (days 70?–?170 of age), the pigs were housed in single boxes. Both, the weaning and the grower/finishing performances (daily body weight gain, feed intake, feed conversion ratio) were similar in both groups. No differences were found between the groups in carcass composition (percentages of cuts, tissues, and protein/fat), and meat quality of pigs. Moreover, the IGF-1R mRNA expression in longissimus muscle was not influenced by the kind of soy product. However, circulating levels of isoflavones were clearly different between pigs fed SBM (genistein 239?±?44; daidzein 162?±?42; equol 12?±?4?ng/ml plasma) and animals fed SPC (genistein 22?±?9 and daidzein 8?±?3, and equol 10?±?3?ng/ml plasma). The results confirm the expected differences in the bioavailability of soy isoflavones, yet, there were no significant differences in performance of pigs fed either soy bean meal or soy protein concentrate.     

A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application

Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl₂, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.     

Behavioural Risk Factors in Mid-Life Associated with Successful Ageing,Disability, Dementia and Frailty in Later Life: A Rapid Systematic Review

Background

Smoking, alcohol consumption, poor diet and low levels of physical activity significantly contribute to the burden of illness in developed countries. Whilst the links between specific and multiple risk behaviours and individual chronic conditions are well documented, the impact of these behaviours in mid-life across a range of later life outcomes has yet to be comprehensively assessed. This review aimed to provide an overview of behavioural risk factors in mid-life that are associated with successful ageing and the primary prevention or delay of disability, dementia, frailty and non-communicable chronic conditions.

Methods

A literature search was conducted to identify cohort studies published in English since 2000 up to Dec 2014. Multivariate analyses and a minimum follow-up of five years were required for inclusion. Two reviewers screened titles, abstracts and papers independently. Studies were assessed for quality. Evidence was synthesised by mid-life behavioural risk for a range of late life outcomes.

Findings

This search located 10,338 individual references, of which 164 are included in this review. Follow-up data ranged from five years to 36 years. Outcomes include dementia, frailty, disability and cardiovascular disease. There is consistent evidence of beneficial associations between mid-life physical activity, healthy ageing and disease outcomes. Across all populations studied there is consistent evidence that mid-life smoking has a detrimental effect on health. Evidence specific to alcohol consumption was mixed. Limited, but supportive, evidence was available relating specifically to mid-life diet, leisure and social activities or health inequalities.

Conclusions

There is consistent evidence of associations between mid-life behaviours and a range of late life outcomes. The promotion of physical activity, healthy diet and smoking cessation in all mid-life populations should be encouraged for successful ageing and the prevention of disability and chronic disease.     

The C-terminal domain of TPX2 is made of alpha-helical tandem repeats

Background

TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains.

Methods

We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements.

Results

Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila).

Conclusions

Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.