Local structural preferences in shaping tau amyloid polymorphism.

Tauopathies encompass a group of neurodegenerative disorders characterised by diverse tau amyloid fibril structures. The persistence of polymorphism across tauopathies suggests that distinct pathological conditions dictate the adopted polymorph for each disease. However, the extent to which intrinsic structural tendencies of tau amyloid cores contribute to fibril polymorphism remains uncertain. Using a combination of experimental approaches, we here identify a new amyloidogenic motif, PAM4 (Polymorphic Amyloid Motif of Repeat 4), as a significant contributor to tau polymorphism. Calculation of per-residue contributions to the stability of the fibril cores of different pathologic tau structures suggests that PAM4 plays a central role in preserving structural integrity across amyloid polymorphs. Consistent with this, cryo-EM structural analysis of fibrils formed from a synthetic PAM4 peptide shows that the sequence adopts alternative structures that closely correspond to distinct disease-associated tau strains. Furthermore, in-cell experiments revealed that PAM4 deletion hampers the cellular seeding efficiency of tau aggregates extracted from Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy patients, underscoring PAM4's pivotal role in these tauopathies. Together, our results highlight the importance of the intrinsic structural propensity of amyloid core segments to determine the structure of tau in cells, and in propagating amyloid structures in disease.

Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid.

α-, ß-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson's disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member.

Structural evolution of fibril polymorphs during amyloid assembly.

Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.

Towards sustainable Cleaning-in-Place (CIP) in dairy processing: Exploring enzyme-based approaches to cleaning in the Cheese industry.

Cleaning-in-place (CIP) is the most commonly used cleaning and sanitation system for processing lines, equipment, and storage facilities such as milk silos in the global dairy processing industry. CIP employs thermal treatments and nonbiodegradable chemicals (acids and alkalis), requiring appropriate neutralization before disposal, resulting in sustainability challenges. In addition, biofilms are a major source of contamination and spoilage in dairy industries, and it is believed that current chemical CIP protocols do not entirely destroy biofilms. Use of enzymes as effective agents for CIP and as a more sustainable alternative to chemicals and thermal treatments is gaining interest. Enzymes offer several advantages when used for CIP, such as reduced water usage (less rinsing), lower operating temperatures resulting in energy savings, shorter cleaning times, and lower costs for wastewater treatment. Additionally, they are typically derived from natural sources, are easy to neutralize, and do not produce hazardous waste products. However, even with such advantages, enzymes for CIP within the dairy processing industry remain focused mainly on membrane cleaning. Greater adoption of enzyme-based CIP for cheese industries is projected pending a greater knowledge relating to cost, control of the process (inactivation kinetics), reusability of enzyme solutions, and the potential for residual activity, including possible effects on the subsequent product batches. Such studies are essential for the cheese industry to move toward more energy-efficient and sustainable cleaning solutions.

The in-tissue molecular architecture of ß-amyloid pathology in the mammalian brain.

Amyloid plaques composed of Aß fibrils are a hallmark of Alzheimer's disease (AD). However, the molecular architecture of amyloid plaques in the context of fresh mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the in situ molecular architecture of Aß fibrils in the AppNL-G-F familial AD mouse model containing the Arctic mutation and an atomic model of ex vivo purified Arctic Aß fibrils. We show that in-tissue Aß fibrils are arranged in a lattice or parallel bundles, and are interdigitated by subcellular compartments, extracellular vesicles, extracellular droplets and extracellular multilamellar bodies. The Arctic Aß fibril differs significantly from an earlier AppNL-F fibril structure, indicating a striking effect of the Arctic mutation. These structural data also revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises ß-amyloid plaque pathology.

Disease-relevant ß2-microglobulin variants share a common amyloid fold.

ß2-microglobulin (ß2m) and its truncated variant ΔΝ6 are co-deposited in amyloid fibrils in the joints, causing the disorder dialysis-related amyloidosis (DRA). Point mutations of ß2m result in diseases with distinct pathologies. ß2m-D76N causes a rare systemic amyloidosis with protein deposited in the viscera in the absence of renal failure, whilst ß2m-V27M is associated with renal failure, with amyloid deposits forming predominantly in the tongue. Here we use cryoEM to determine the structures of fibrils formed from these variants under identical conditions in vitro. We show that each fibril sample is polymorphic, with diversity arising from a 'lego-like' assembly of a common amyloid building block. These results suggest a 'many sequences, one amyloid fold' paradigm in contrast with the recently reported 'one sequence, many amyloid folds' behaviour of intrinsically disordered proteins such as tau and Aß.

Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein.

Αlpha-synuclein (αS) is an intrinsically disordered protein which exhibits a high degree of conformational heterogeneity. In vivo, αS experiences various environments which cause adaptation of its structural ensemble. Divalent metal ions are prominent in synaptic terminals where αS is located and are thought to bind to the αS C-terminal region. Herein, we used native nanoelectrospray ionization ion mobility-mass spectrometry to investigate changes in the charge state distribution and collision cross sections of wild-type N-terminally acetylated (NTA) αS, along with a deletion variant (ΔΔNTA) which inhibits amyloid formation and a C-terminal truncated variant (119NTA) which increases the rate of amyloid formation. We also examine the effect of the addition of divalent metal ions, Ca2+, Mn2+, and Zn2+, and correlate the conformational properties of the αS monomer with the ability to aggregate into amyloid, measured using Thioflavin T fluorescence and negative stain transmission electron microscopy. We find a correlation between the population of species with a low collision cross section and accelerated amyloid assembly kinetics, with the presence of metal ions resulting in protein compaction and causing ΔΔ to regain its ability to form an amyloid. The results portray how the αS conformational ensemble is governed by specific intramolecular interactions that influence its amyloidogenic behavior.

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub.

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

Enzymatic Disruption of Biofilms During Cheese Manufacturing: A Mini Review.

Bacteria are capable of colonizing industrial processing surfaces creating biofilms on them which may adversely affect the quality and safety of products. Traditional cleaning-in-place (CIP) treatments using caustic and nitric acid solutions have been known to exhibit variable efficiency in eliminating biofilm bacteria. Here, we introduce enzymes as an alternative to traditional CIP treatments and discuss their mechanism of action against bacterial biofilms in cheese manufacturing. In addition, we discuss research gaps namely thermal stability, substrate specificity and residual activity of enzymes that may play a vital role in the selection of enzymes with optimal effectiveness against multi species biofilms. The outcome of this mini review will aid in the development of a novel and sustainable enzyme-based CIP treatment during cheese manufacturing in the future.

Invited review: Starter lactic acid bacteria survival in cheese: New perspectives on cheese microbiology.

The importance of starter cultures to cheese manufacture and ripening is well known. Starters are inoculated into cheese milk at a level of ∼106 cfu/mL either from a bulk culture or using commercial direct-to-vat cultures. Before ripening, starters grow in the milk to reach populations of 107 to 109 cfu/g of curd depending on processing variables such as cook temperature, inclusion of washing steps, degree of partitioning with curds and whey, and importantly salt addition rate. Inherent strain-related properties also determine final populations in the curd following manufacture and include temperature sensitivity, salt sensitivity, presence of prophage, autolytic and permeabilization properties (which are influenced by processing steps), presence and type of cell envelope proteinase, and metabolic activity. Ripening of important industrial cheese varieties such as Cheddar, Dutch, Swiss, and Italian-type cheese varieties is characterized by extended storage under temperature-controlled conditions enabling characteristic flavor and texture development to occur. Over ripening, microbiological, biochemical and enzymatic changes occur with a decline in starter viability, release of intracellular enzymes, hydrolysis of proteins, carbohydrates and lipids, and formation of a range of volatile and nonvolatile flavor components. Recent reports suggest that starter strains may be present during the later stages of ripening and therefore their potential role needs to be reconsidered. This review will focus on our current understanding of starter viability and vitality during cheese ripening and will also review the area of starter permeabilization, autolysis, and enzyme release.

Gaps in the assortment of rapid assays for microorganisms of interest to the dairy industry.

This review presents the results of a study into the offering of rapid microbial detection assays to the Irish dairy industry. At the outset, a consultation process was undertaken whereby key stakeholders were asked to compile a list of the key microorganisms of interest to the sector. The resultant list comprises 19 organisms/groups of organisms divided into five categories: single pathogenic species (Cronobacter sakazakii, Escherichia coli and Listeria monocytogenes); genera containing pathogenic species (Bacillus, Clostridium, Listeria, Salmonella; Staphylococcus); broad taxonomic groupings (Coliforms, Enterobacteriaceae, fecal Streptococci, sulfite reducing bacteria/sulfite reducing Clostridia [SRBs/SRCs], yeasts and molds); organisms displaying certain growth preferences or resistance as regards temperature (endospores, psychrotrophs, thermodurics, thermophiles); indicators of quality (total plate count, Pseudomonas spp.). A survey of the rapid assays commercially available for the 19 organisms/groups of organisms was conducted. A wide disparity between the number of rapid tests available was found. Four categories were used to summarize the availability of rapid assays per organism/group of organisms: high coverage (>15 assays available); medium coverage (5-15 assays available); low coverage (<5 assays available); no coverage (0 assays available). Generally, species or genera containing pathogens, whose presence is regulated-for, tend to have a good selection of commercially available rapid assays for their detection, whereas groups composed of heterogenous or even undefined genera of mainly spoilage organisms tend to be "low coverage" or "no coverage." Organisms/groups of organisms with "low coverage" by rapid assays include: Clostridium spp.; fecal Streptococci; and Pseudomonas spp. Those with "no coverage" by rapid assays include: endospores; psychrotrophs; SRB/SRCs; thermodurics; and thermophiles. An important question is: why have manufacturers of rapid microbiological assays failed to respond to the necessity for rapid methods for these organisms/groups of organisms? The review offers explanations, ranging from the technical difficulty involved in detecting as broad a group as the thermodurics, which covers the spores of multiple sporeforming genera as well at least six genera of mesophilic nonsporeformers, to the taxonomically controversial issue as to what constitutes a fecal Streptococcus or SRBs/SRCs. We review two problematic areas for assay developers: validation/certification and the nature of dairy food matrices. Development and implementation of rapid alternative test methods for the dairy industry is influenced by regulations relating to both the microbiological quality standards and the criteria alternative methods must meet to qualify as acceptable test methods. However, the gap between the certification of developer's test systems as valid alternative methods in only a handful of representative matrices, and the requirement of dairy industries to verify the performance of alternative test systems in an extensive and diverse range of dairy matrices needs to be bridged before alternative methods can be widely accepted and adopted in the dairy industry. This study concludes that many important dairy matrices have effectively been ignored by assay developers.

Protein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent.

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.

A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair.

The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.

Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis.

The effects of heat and chemical treatments on Staphylococcus aureus viability and physiology and their subsequent effects on antibody binding ability and cell morphology were measured. Treatments included lethal and sublethal heat; exposure to organic acids, salt, and sodium hydroxide; and freeze-thawing. Strain-related differences in viability were noted depending on treatment and were reflected in changes in physiology as monitored by flow cytometry (FCM) using three different staining protocols: SYTO 9/propidium iodide (PI), DiOC2(3), or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments that resulted in significant losses in viability as measured by plate counting were reflected better by the first two staining combinations, as intracellular calcein-AM uptake may have been impaired by certain treatments. FCM analysis using labeling by commercial anti-S. aureus antibodies indicated that differences in cell physiology as a result of treatments influenced immunofluorescence detection. The ratio of the mean fluorescence intensities of stained cells to those of unstained cells [MFI/MFI(us)] varied with treatment, five of these treatments, including freeze-thaw, citric acid, oxalic acid, NaCl, and NaOH treatments, resulted in significantly lower fluorescence values compared to controls.IMPORTANCE FCM data indicated that cells conventionally considered to be dead and which would not give rise to CFU in a plate count assay, e.g., cells heated to 80°C, were labeled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye, these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physicochemical treatment were strain related, reflecting the complexity of the phenomenon under study and illustrating that substantial validation of any new antibody detection-based method, including physiological staining and cell sorting, should be undertaken. Researchers should be aware of physicochemical treatments causing false-negative results: in this study, freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy carried out on treated cells revealed a range of morphological changes resulting from physicochemical treatments which may have hindered antibody binding.

Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

Structure and dynamics of the yeast SWR1-nucleosome complex.

The yeast SWR1 complex exchanges histone H2A in nucleosomes with Htz1 (H2A.Z in humans). The cryo-electron microscopy structure of the SWR1 complex bound to a nucleosome at 3.6-angstrom resolution reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at superhelical location 2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to SWR1 complex. The unwrapping, as monitored by single-molecule data, is stabilized and has its dynamics altered by adenosine triphosphate binding but does not require hydrolysis.

Structure and regulation of the human INO80-nucleosome complex.

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

Application of Flow Cytometry to the Detection of Pathogenic Bacteria.

Outbreaks of infections have emphasized the necessity for rapid and economic detection methods for pathogens in samples ranging from those of clinical origin to food products during production and retail storage, and increasingly, in environmental samples. Flow cytometry (FCM) allows the rapid acquisition of multi-parametric data regarding cell populations within fluidised samples. However, the application of FCM to pathogen detection depends on the availability of specific fluorescent probes such as antibodies and RNA probes capable of detecting and isolating pathogens from these diverse samples. A particular issue for FCM methodology is the ability to recover and discriminate bacteria from the sample matrix which may pose a major technical hurdle towards accurate and sensitive analysis. This review article focuses on detection of pathogens using FCM in samples originating from food, water, environmental and clinical sources and outlines the current state of the art and potential future applications.