Search

• New structures have shown that there is a remarkable diversity and complexity of the amyloid fold. • The same sequence forms different amyloid structures in different diseases. • Polymorph selection in vivo is likely dependent on cellular and extracellular constraints. • Locally disordered regions in amyloid fibrils are functionally and structurally important. • Ligand binding, post-translational modifications and non-protein components contribute to the structural diversity of the amyloid fold. Solving the amyloid puzzle requires an integrated use of structural and functional approaches. In recent years our understanding of amyloid structure has been revolutionised by innovations in cryo-electron microscopy, electron diffraction and solid-state NMR. These techniques have yielded high-resolution structures of fibrils isolated from patients with neurodegenerative disease, as well as those formed from amyloidogenic proteins in vitro. The results not only show the expected cross-β amyloid structure, but also reveal that the amyloid fold is unexpectedly diverse and complex. Here, we discuss this diversity, highlighting dynamic regions, ligand binding motifs, cavities, non-protein components, and structural polymorphism. Collectively, these variations combine to allow the generic amyloid fold to be realised in three dimensions in different ways, and this diversity may be related to the roles of fibrils in disease. [ABSTRACT FROM AUTHOR]

The application of RNA sequencing in commercial poultry could facilitate a novel approach toward food safety with respect to identifying conditions in food production that mitigate transcription of genes associated with virulence and survivability. In this study, we evaluated the effects of disinfectant exposure on the transcriptomes of two field isolates of Salmonella Heidelberg (SH) isolated from a commercial broiler processing plant in 1992 and 2014. The isolates were each exposed separately to the following disinfectants commonly used in poultry processing: cetylpyridinium chloride (CPC), acidified calcium hypochlorite (aCH), and peroxyacetic acid (PAA). Exposure times were 8 s with CPC to simulate a poultry processing dipping station or 90 min with aCH and PAA to simulate the chiller tank in a poultry processing plant at 4°C. Based on comparison with a publicly available annotated SH reference genome with 5,088 genes, 90 genes were identified as associated with virulence, pathogenicity, and resistance (VPR). Of these 90 VPR genes, 9 (10.0%), 28 (31.1%), and 1 (1.1%) gene were upregulated in SH 2014 and 21 (23.3%), 26 (28.9%), and 2 (2.2%) genes were upregulated in SH 2014 challenged with CPC, aCH, and PAA, respectively. This information and previously reported MICs for the three disinfectants with both SH isolates allow researchers to make more accurate recommendations regarding control methods of SH and public health considerations related to SH in food production facilities where SH has been isolated. For example, the MICs revealed that aCH is ineffective for SH inhibition at regulatory levels allowed for poultry processing and that aCH was ineffective for inhibiting SH growth and caused an upregulation of VPR genes. [ABSTRACT FROM AUTHOR]

Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 µm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease. [ABSTRACT FROM AUTHOR]

Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid β (Aβ) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aβ in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aβ properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aβ production in the A2T mutant along with an enhancedAβ generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aβ42 peptide aggregation, dramatically change the properties of the Aβ40 pool with A2V accelerating and A2T delaying aggregation of the Aβ peptides. In line with the kinetic data, Aβ A2Tdemonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aβ40. We propose that in addition to the reduced β-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously. [ABSTRACT FROM AUTHOR]

Abstract: Aims: Patients with atrioventricular (AV) block can develop left ventricular (LV) dysfunction with long-term right ventricular pacing (RVP). We investigated the role of RVP-induced LV dyssynchrony in this adverse remodeling. Methods and Results: Nineteen patients with normal LV function undergoing pacemaker implantation for AV block were included. Right ventricular pacing leads were positioned at the apex. Two-dimensional and tissue Doppler echocardiography was performed before and immediately after implantation and at the end of follow-up. The maximal delay between peak velocities of opposing basal LV walls was measured using tissue Doppler echocardiography, as an index of LV dyssynchrony. With the initiation of RVP, LV dyssynchrony increased in some patients and decreased in others, as compared with intrinsic rhythm. The RVP-induced change in dyssynchrony inversely correlated with baseline dyssynchrony (r = −0.686, P = .010). After 28 ± 3.6 months, LV end-systolic volume (ESV) increased, and ejection fraction decreased (from 34 ± 12 to 40 ± 20 mL, P = .010 and from 65% ± 6% to 56% ± 11%, P < .001, respectively). The change in LV ESV was greater in patients with 60% or greater cumulative RVP (9.9 vs 0.08 mL, P = .027). Within this frequently paced group, the RVP-induced change in dyssynchrony correlated with the increase in LV ESV (r = 0.727, P = .026). Patients who had a 15% or greater increase in LV ESV had greater RVP-induced change in dyssynchrony (28.4 vs −7.8 milliseconds, P = .037). Conclusion: Some patients with AV block experience an increase in LV dyssynchrony with RVP. Increased LV dyssynchrony predicts adverse LV remodeling during long-term follow-up. [Copyright &y& Elsevier]

The amyloid-like aggregation propensity present in most globular proteins is generally considered to be a secondary side effect resulting from the requirements of protein stability. Here, we demonstrate, however, that mutations in the globular and amyloid state are thermodynamically correlated rather than simply associated. In addition, we show that the standard genetic code couples this structural correlation into a tight evolutionary relationship. We illustrate the extent of this evolutionary entanglement of amyloid propensity and globular protein stability. Suppressing a 600-Ma-conserved amyloidogenic segment in the p53 core domain fold is structurally feasible but requires 7-bp substitutions to concomitantly introduce two aggregation-suppressing and three stabilizing amino acid mutations. We speculate that, rather than being a corollary of protein evolution, it is equally plausible that positive selection for amyloid structure could have been a driver for the emergence of globular protein structure. • Mutations in the globular and amyloid state are thermodynamically correlated • The genetic code tightens this relationship between the amyloid and native state • Strongly amyloidogenic sequences in globular proteins are deeply conserved • Positive selection of amyloid propensity will favor globular protein stability Langenberg et al. show that amyloid propensity favors protein stability. This results from the energetic correlation of mutation in the native and amyloid state. The genetic code tightens this relationship so that stable amyloidogenic sequences are deeply conserved. Positive selection of amyloidogenic sequences could therefore have favored the evolution of globular structure. [ABSTRACT FROM AUTHOR]

Prions of lower eukaryotes are self-templating protein aggregates with cores formed by parallel in-register beta strands. Short aggregation-prone glutamine (Q)- and asparagine (N)-rich regions embedded in longer disordered domains have been proposed to act as nucleation sites that initiate refolding of soluble prion proteins into highly ordered fibrils, termed amyloid. We demonstrate that a short Q/N-rich peptide corresponding to a proposed nucleation site in the prototype Saccharomyces cerevisiae prion protein Sup35 is sufficient to induce infectious cytosolic prions in mouse neuroblastoma cells ectopically expressing the soluble Sup35 NM prion domain. Embedding this nucleating core in a non-native N-rich sequence that does not form amyloid but acts as an entropic bristle quadruples seeding efficiency. Our data suggest that large disordered sequences flanking an aggregation core in prion proteins act as not only solubilizers of the monomeric protein but also breakers of the formed amyloid fibrils, enhancing infectivity of the prion seeds. • A short peptide derived from Sup35 (p103–113) forms rigid amyloid fibrils • p103–113 fibrils can induce infectious Sup35 NM prions in mammalian cells • Embedding p103–113 in an N-rich sequence increases fibril brittleness • Increased fibril brittleness enhances prion-inducing capacity A protein aggregate can contain infective properties, prions being the prime example. Michiels et al. show that these infective properties are encoded in the specific amino acid sequence flanking the aggregation core of a protein. These so-called entropic bristle sequences drive fiber brittleness, a crucial feature for amyloid infectivity. [ABSTRACT FROM AUTHOR]

• Increase in moisture resulted in the increased survival of S. Typhimurium in poultry litter. • Increase in the moisture content reduced the effect of ozone in eliminating pathogens. • Extended exposure time resulted in increase in pathogen inactivation. • Low ozone concentrations (<6%) and low exposure time was not effective in pathogen inactivation in poultry litter. • Gaseous O3 treatment has a potential to be used as decontamination technique for animal wastes. Poultry litter is used as soil amendment or organic fertilizer. While poultry litter is enriched with organic matter suitable for land, the presence of pathogens such as Salmonella in poultry litter is a concern. To investigate the effect of gaseous ozone on pathogen reductions in poultry litter, this study conducted a series of experiments that involved understanding of Salmonella Typhimurium and Escherichia coli O157:H7 inactivation at various doses of Ozone (O 3) in wet and dry poultry litter conditions. Previously, ozone treatment has been shown to disinfect the surface of foods and plant materials including fruits, juices, and wastewater, however, additional research are needed to better understand the impacts of ozone on treatment of soil amendments. Sanitizing methods capable of eliminating pathogens of soil amendments are crucial to mitigate disease outbreaks related with litter/manure-based fertilizers. In this study, a bench scale continuous ozone treatment system was designed to produce O 3 gas, with a range O 3 concentrations (7.15–132.46 mg·L−1), monitor ozone concentrations continuously, and control the ozone exposure time (15 to 90 mins) to understand the effectiveness of O 3 in eliminating S. Typhimurium and E. coli O157:H7 in poultry litter. Results showed that 7.15 mg·L-1 did not reduce the counts of S. Typhimurium until exposure to O 3 for 90 min. The O 3 concentrations of 43.26 ~ 132.46 mg·L-1 exposure reduced the bacterial counts. Furthermore, the moisture content of poultry litter was found to be an influencing factor for pathogen reduction. The pathogen reduction rates were reduced when the moisture content was increased. At higher moisture content, high concentrations of O 3 (132.46 mg·L-1) were needed for pathogen reductions. The moisture content of 30% or lower was found to be more effective for controlling pathogen levels in poultry litter. Our study demonstrates that gaseous O 3 treatment could be used as an additional decontamination technique to ensure the certain degree of microbiological safety of poultry litter based soil amendment. [ABSTRACT FROM AUTHOR]

Vaccines against virulent Newcastle disease virus (NDV) are widely available and can be protective, but improved vaccination protocols are needed to prevent clinical disease and reduce virus circulation. The present study evaluated the efficacy of two commercial vaccines alone or in combination: a live attenuated NDV vaccine (LV) and a recombinant herpesvirus of turkeys vector expressing the fusion protein of NDV and the virus protein 2 of infectious bursal disease virus (rHVT-ND-IBD). Chickens were vaccinated with one of four vaccination protocols: live vaccine (LV) at 1 and 11 days of age (DOA), rHVT ND-IBD and LV at 1 DOA, rHVT ND-IBD at 1 DOA boosted with an LV at 11 DOA, and rHVT ND-IBD at 1 DOA. The vaccinated birds were challenged at different time points (3 or 4 weeks of age) with the California 2018 virus. The mortality, clinical signs, mean death time (MDT), humoral response before and after vaccination, and virus shedding after challenge were evaluated. All vaccination protocols were able to prevent mortality, reduce virus shedding, and induce antibody levels before the challenge at 3 and 4 weeks-old. Overall, the antibody levels before the challenge at 4 weeks were significantly higher in all groups vaccinated with the rHVT ND-IBD when compared to levels in 3 week old birds. The combination of recombinant rHVT ND-IBD with a live vaccine at one-day-old seems to be a better combination, due to the absence of clinical signs, higher antibody levels pre and post-challenge, and reduced virus shedding at any time point after the challenge at 3 or 4 weeks of age with the California 2018 virus. [ABSTRACT FROM AUTHOR]

The disposal of by-products, such as poultry litter, and carcasses is a serious issue because of risks associated with microbial pathogens, and controlling the pathogen risks requires identifying improved pre-treatment methods capable of inactivating pathogens. As poultry litter and carcasses are known to be major reservoirs of pathogenic microorganisms such as Salmonella and Escherichia coli (E. coli), improvement in existing understanding of the inactivation of these pathogens in poultry litter and carcasses is needed to determine the effective treatment time and temperature. Here we conducted a study to assess the thermal inactivation of 2 common bacteria in poultry productive systems: Escherichia coli (E. coli) and Salmonella enterica (Salmonella). The inactivation study was conducted at 50°C and 60°C using 3 different feedstocks: (1) poultry carcasses, (2) poultry litter, and (3) mixture of poultry litter and carcasses. Each feedstock was inoculated with known concentrations of E. coli and Salmonella prior to thermophilic digestion experiments at 50°C and 60°C. Regardless of feedstock types, E. coli survival was extended beyond 3 d at 50°C. In contrast, Salmonella was no longer detectable within 3 d at 50°C. At 60°C, both E. coli and Salmonella were undetected within an hour. There was no significant difference (at P < 0.05) in pathogen survival among 3 feedstocks. [ABSTRACT FROM AUTHOR]

Live virus vaccines are commonly used in poultry production, particularly in broilers. Massive application and generation of a protective local mucosal and humoral immunity with no adverse effects is the main goal for this strategy. Live virus vaccines can be improved by adding adjuvants to boost mucosal innate and adaptive responses. In a previous study we showed that diatomaceous earth (DE) can be used as adjuvant in inactivated vaccines. The aim of this study was to test DE as adjuvant in an Ark-DPI live infectious bronchitis virus (IBV) vaccine after ocular or spray application. Titrating the virus alone or after addition of DE showed that DE had no detrimental effect on the vaccine virus. However, adding DE to the vaccine did not induce higher IgG titers in the serum and IgA titers in tears. It also did not affect the frequency of CD4+ T cells, CD8+ T cells and monocytes/macrophages in the blood and the spleen determined by flow cytometry. In addition, protection generated against IBV homologous challenges, measured by viral load in tears, respiratory signs and histopathology in tracheas, did not vary when DE was present in the vaccine formulation. Finally, we confirmed through our observations that Ark vaccines administered by hatchery spray cabinet elicit weaker immune responses and protection against an IBV homologous challenge compared to the same vaccine delivered via ocular route. [ABSTRACT FROM AUTHOR]

Wild-type avian encephalomyelitis virus (AEV) causes neurological signs in young chicks but no disease in pullets after oral or intracutaneous infection. However, if the virus gets embryo-adapted by serial passaging in chicken embryos, it will cause AE after intracutaneous infection in chickens of all ages. Recently, several cases of AE in layer pullets occurring shortly after intracutaneous vaccination were described. The present investigation was initiated to determine if vaccines that had inadvertently been embryo-adapted were responsible for these outbreaks. Virus isolation was done from two vaccines and one field sample. One of the vaccines had been used in one of the flocks before the outbreak. After the first passage, regardless of the inoculum, no embryo was paralyzed, indicating that the vaccines and the field isolate were not embryo-adapted. After seven passages all three strains were fully embryo-adapted causing typical lesions in the embryos. Viral load as determined by RT-qPCR remained constant during the passages. Partial sequences of the VP2 gene of vaccines, the field sample and four other field isolates were nearly identical and highly similar to published sequences from all over the world; only sequences originating from non-vaccinated birds were clearly set apart. Analysis of whole genomes identified two single nucleotide polymorphisms (SNPs) that distinguished wild-type and embryo-adapted strains. Sanger sequencing brains and nerves of the five field isolates and of the first, third and fifth passages of the isolates showed that the mutations indicating embryo-adaptation were first observed in the fifth passage. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to determine whether a multivalent modified-live virus vaccine containing noncytopathic bovine viral diarrhea virus (BVDV) administered off-label to pregnant cattle can result in persistently infected fetuses and to assess whether vaccinal strains can be shed to unvaccinated pregnant cattle commingling with vaccinates. Nineteen BVDV-naïve pregnant heifers were randomly assigned to two groups: cattle vaccinated near Day 77 of gestation with modified-live virus vaccine containing BVDV-1a (WRL strain), bovine herpes virus-1, parainfluenza 3, and bovine respiratory syncytial virus (Vx group; N = 10) or control unvaccinated cattle (N = 9). During the course of the study a voluntary stop-sale/recall was conducted by the manufacturer because of the presence of a BVDV contaminant in the vaccine. At Day 175 of gestation, fetuses were removed by Cesarean section and fetal tissues were submitted for virus isolation, and quantitative reverse transcription polymerase chain reaction using BVDV-1- and BVDV-2-specific probes. Nucleotide sequencing of viral RNA was performed for quantitative reverse transcription polymerase chain reaction-positive samples. Two vaccinated and two control heifers aborted their pregnancies, but their fetuses were unavailable for BVDV testing. Virus was isolated from all eight fetuses in the Vx group heifers and from 2 of 7 fetuses in the control unvaccinated heifers. Only BVDV-2 was detected in fetuses from the Vx group, and only BVDV-1 was detected in the two fetuses from the control group. Both BVDV-1 and BVDV-2 were detected in the vaccine. In conclusion, vaccination of pregnant heifers with a contaminated modified-live BVDV vaccine resulted in development of BVDV-2 persistently infected fetuses in all tested vaccinated animals. Furthermore, BVDV was apparently shed to unvaccinated heifers causing fetal infections from which only BVDV-1 was detected. [Copyright &y& Elsevier]

Fabry disease is a lysosomal storage disorder caused by loss of α-galactosidase function. More than 500 Fabry disease mutants have been identified, the majority of which are structurally destabilized. A therapeutic strategy under development for lysosomal storage diseases consists of using pharmacological chaperones to stabilize the structure of the mutant protein, thereby promoting lysosomal delivery over retrograde degradation. The substrate analog 1-deoxygalactonojirimycin (DGJ) has been shown to restore activity of mutant α-galactosidase and is currently in clinical trial for treatment of Fabry disease. However, only ∼65% of tested mutants respond to treatment in cultured patient fibroblasts, and the structural underpinnings of DGJ response remain poorly explained. Using computational modeling and cell culture experiments, we show that the DGJ response is negatively affected by protein aggregation of α-galactosidase mutants, revealing a qualitative difference between misfolding-associated and aggregation-associated loss of function. A scoring function combining predicted thermodynamic stability and intrinsic aggregation propensity of mutants captures well their aggregation behavior under overexpression in HeLa cells. Interestingly, the same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression levels as well. Our observations reinforce the idea that treatment of aggregation-associated loss of function observed for the more severe α-galactosidase mutants could be enhanced by combining pharmacological chaperone treatment with the suppression of mutant aggregation, e.g. via proteostatic regulator compounds that increase cellular chaperone expression. [ABSTRACT FROM AUTHOR]

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-merle state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein. [ABSTRACT FROM AUTHOR]