Glycation affects fibril formation of Aß peptides.

Increasing evidence shows that ß-amyloid (Aß) peptides, which are associated with Alzheimer disease (AD), are heavily glycated in patients, suggesting a role of this irreversible nonenzymatic post-translational modification in pathology. Previous reports have shown that glycation increases the toxicity of the Aß peptides, although little is known about the mechanism. Here, we used the natural metabolic by-product methylglyoxal as a glycating agent and exploited various spectroscopic methods and atomic force microscopy to study how glycation affects the structures of the Aß40 and Aß42 peptides, the aggregation pathway, and the morphologies of the resulting aggregates. We found that glycation significantly slows down but does not prevent ß-conversion to mature fibers. We propose that the previously reported higher toxicity of the glycated Aß peptides could be explained by a longer persistence in an oligomeric form, usually believed to be the toxic species.

Quantitative analysis of ezrin turnover dynamics in the actin cortex.

Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.

Atomic force microscopy with nanoscale cantilevers resolves different structural conformations of the DNA double helix.

Structural variability and flexibility are crucial factors for biomolecular function. Here we have reduced the invasiness and enhanced the spatial resolution of atomic force microscopy (AFM) to visualize, for the first time, different structural conformations of the two polynucleotide strands in the DNA double helix, for single molecules under near-physiological conditions. This is achieved by identifying and tracking the anomalous resonance behavior of nanoscale AFM cantilevers in the immediate vicinity of the sample.

Corrigendum: The disease associated Tau35 fragment has an increased propensity to aggregate compared to full-length tau.

[This corrects the article DOI: 10.3389/fmolb.2021.779240.].

Detection and identification of body fluid stains using antibody-nanoparticle conjugates.

Body fluids are considered one of the most important evidence types in forensic casework. The presence and location of blood, semen and saliva can provide crucial information to investigators. Current practice relies on an accurate visual examination followed by the use of presumptive tests to determine the identity of the body fluid type. Further laboratory based tests are required to unequivocally confirm the identity of a stain. Body fluid stains can be difficult to detect with the naked eye, particularly on dark backgrounds and hence vital evidence may be overlooked. Current methods are fluid-type specific, with a separate, and different, test required for each body fluid. The laborious nature of such analysis and the impossibility of being carried out at the crime scene, leads to a delay in the investigation process that could prove detrimental to the solving of the case. Hence, there is a need for sensitive, specific and direct methods which can simultaneously detect, differentiate, and locate human fluids on items of forensic evidence. Here, we describe the preparation of functionalized iron oxide nanoparticles conjugated to antibodies specific to blood and saliva components and their use in detecting small traces against non-contrasting substrates including glass, ceramic tile, paper and black fabric. The advantage of our technique is that it can simultaneously detect blood and saliva and can spatially locate and differentiate these body fluid types. Most importantly, our technology, which exploits the superparamagnetic properties of iron oxide nanoparticles, works in situ with no need to remove the body fluid stains for testing and with no washing steps and does not interfere with downstream DNA profiling. Thus, our technology represents a novel and effective alternative to existing methods.

TiO2 Nanotopography-Driven Osteoblast Adhesion through Coulomb's Force Evolution.

Nanotopography is an effective method to regulate cells' behaviors to improve Ti orthopaedic implants' in vivo performance. However, the mechanism underlying cellular matrix-nanotopography interactions that allows the modulation of cell adhesion has remained elusive. In this study, we have developed novel nanotopographic features on Ti substrates and studied human osteoblast (HOb) adhesion on nanotopographies to reveal the interactive mechanism regulating cell adhesion and spreading. Through nanoflat, nanoconvex, and nanoconcave TiO2 nanotopographies, the evolution of Coulomb's force between the extracellular matrix and nanotopographies has been estimated and comparatively analyzed, along with the assessment of cellular responses of HOb. We show that HObs exhibited greater adhesion and spreading on nanoconvex surfaces where they formed super matured focal adhesions and an ordered actin cytoskeleton. It also demonstrated that Coulomb's force on nanoconvex features exhibits a more intense and concentrated evolution than that of nanoconcave features, which may result in a high dense distribution of fibronectin. Thus, this work is meaningful for novel Ti-based orthopaedic implants' surface designs for enhancing their in vivo performance.

Cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions.

Control of cell-surface interaction is necessary for biomaterial applications such as cell sheets, intelligent cell culture surfaces, or functional coatings. In this paper, we propose the emergent property of cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions. Cell morphology measured through various parameters can indicate ideal candidates for these various applications thus reducing the time taken for the screening and development process. The hypothesis of this study is that there is an optimal cell morphology range for enhanced cell proliferation and migration on the surface of biomaterials. To test the hypothesis, primary porcine dermal fibroblasts (PDF, 3 biological replicates) were cultured on ten different surfaces comprising components of the natural extracellular matrix of tissues. Results suggested an optimal morphology with a cell aspect ratio (CAR) between 0.2 and 0.4 for both increased cell proliferation and migration. If the CAR was below 0.2 (very elongated cell), cell proliferation was increased whilst migration was reduced. A CAR of 0.4+ (rounded cell) favoured cell migration over proliferation. The screening process, when it comes to biomaterials is a long, repetitive, arduous but necessary event. This study highlights the beneficial use of testing the cell morphology on prospective prototypes, eliminating those that do not support an optimal cell shape. We believe that the research presented in this paper is important as we can help address this screening inefficiency through the use of the emergent property of cell morphology. Future work involves automating CAR quantification for high throughput screening of prototypes.

The Disease Associated Tau35 Fragment has an Increased Propensity to Aggregate Compared to Full-Length Tau.

Tau35 is a truncated form of tau found in human brain in a subset of tauopathies. Tau35 expression in mice recapitulates key features of human disease, including progressive increase in tau phosphorylation, along with cognitive and motor dysfunction. The appearance of aggregated tau suggests that Tau35 may have structural properties distinct from those of other tau species that could account for its pathological role in disease. To address this hypothesis, we performed a structural characterization of monomeric and aggregated Tau35 and compared the results to those of two longer isoforms, 2N3R and 2N4R tau. We used small angle X-ray scattering to show that Tau35, 2N3R and 2N4R tau all behave as disordered monomeric species but Tau35 exhibits higher rigidity. In the presence of the poly-anion heparin, Tau35 increases thioflavin T fluorescence significantly faster and to a greater extent than full-length tau, demonstrating a higher propensity to aggregate. By using atomic force microscopy, circular dichroism, transmission electron microscopy and X-ray fiber diffraction, we provide evidence that Tau35 aggregation is mechanistically and morphologically similar to previously reported tau fibrils but they are more densely packed. These data increase our understanding of the aggregation inducing properties of clinically relevant tau fragments and their potentially damaging role in the pathogenesis of human tauopathies.

Synthesis, characterisation and intracellular imaging of PEG capped BEHP-PPV nanospheres.

Aqueous dispersions of poly(ethylene glycol) (PEG) capped poly[2-(2',5'-bis(2''-ethylhexyloxy)phenyl)-1,4-phenylene vinylene] (BEHP-PPV) nanospheres with an average particle diameter of 13 nm have been synthesised by a miniemulsion route and used in simple intracellular imaging experiments.

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization.

Ca2+ signalling to the nucleus is thought to occur by calmodulin entry into the nucleus where calmodulin has many functions. In the present study we have investigated the role of Ca2+ and the N- and C-terminal lobes of calmodulin in its subnuclear targeting by using fluorescently labelled calmodulin and its mutants and confocal microscopy. Our data show, first, that Ca2+ stimulation induces a reorganization of subnuclear structures to which apo-calmodulin can bind. Secondly, Ca2+-independent association of the C-terminal lobe is seen with subnuclear structures such as chromatin, the nuclear envelope and the nucleoli. Thirdly, Ca2+-dependent accumulation of both calmodulin and the C-terminal calmodulin lobe occurs in the nucleoli. The N-terminal lobe of calmodulin does not show significant binding to subnuclear structures although, similarly to the C-terminal lobe, it accumulates in the nucleoplasm of wheat germ agglutinin-blocked nuclei suggesting that a facilitated nuclear export mechanism exists for calmodulin.

The RNA-Recognition Motifs of TAR DNA-Binding Protein 43 May Play a Role in the Aberrant Self-Assembly of the Protein.

The TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein implicated in gene regulation and RNA processing and shuffling. It is a ribonuclear protein that carries out most of its functions by binding specific nucleic acid sequences with its two RNA-recognition motifs, RRM1 and RRM2. TDP-43 has been identified in toxic cytosolic inclusions in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). The unstructured C-terminus has prion-like behavior and has been considered the driver of the aberrant self-assembly of TDP-43. In this work, we set out to test the hypothesis that the RNA-binding domains could also play a role in protein aggregation. This knowledge could be of important value for understanding TDP-43 aberrant, disease-leading behavior and, in the future, inform the design of small molecules that could prevent or slow down protein aggregation by exploiting the RNA-binding properties of the protein. We investigated the behavior of the two tandem RRM domains separately and linked together and studied their self-assembly properties and RNA-binding ability with a number of biophysical techniques. The picture that emerges from our study suggests that this region of the protein plays an important and so far unexplored role in the aggregation of this protein.

Mucoadhesion of Progesterone-Loaded Drug Delivery Nanofiber Constructs.

Mucoadhesive delivery systems have attracted remarkable interest recently, especially for their potential to prolong dosage form resident times at sites of application such as the vagina or nasal cavity, thereby improving convenience and compliance as a result of less frequent dosage. Mucoadhesive capabilities need to be routinely quantified during the development of these systems. This is however logistically challenging due to difficulties in obtaining and preparing viable mucosa tissues for experiments. Utilizing artificial membranes as a suitable alternative for quicker and easier analyses of mucoadhesion of these systems is currently being explored. In this study, the mucoadhesive interactions between progesterone-loaded fibers (with varying carboxymethyl cellulose (CMC) content) and either artificial (cellulose acetate) or mucosa membranes are investigated by texture analysis and results across models are compared. Mucoadhesion to artificial membrane was about 10 times that of mucosa, though statistically significant ( p = 0.027) association between the 2 data sets was observed. Furthermore, a hypothesis relating fiber-mucosa interfacial roughness (and unfilled void spaces on mucosa) to mucoadhesion, deduced from some classical mucoadhesion theories, was tested to determine its validity. Points of interaction between the fiber and mucosa membrane were examined using atomic force microscopy (AFM) to determine the depths of interpenetration and unfilled voids/roughness, features crucial to mucoadhesion according to the diffusion and mechanical theories of mucoadhesion. A Kendall's tau and Goodman-Kruskal's gamma tests established a monotonic relationship between detaching forces and roughness, significant with p-values of 0.014 and 0.027, respectively. A similar relationship between CMC concentration and interfacial roughness was also confirmed. We conclude that AFM analysis of surface geometry following mucoadhesion can be explored for quantifying mucoadhesion as data from interfacial images correlates significantly with corresponding detaching forces, a well-established function of mucoadhesion.

Studies of G-quadruplexes formed within self-assembled DNA mini-circles.

We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.

A phenomenological density-scaling approach to lamellipodial actin dynamics(†).

The integration of protein function studied in vitro in a dynamic system like the cell lamellipodium remains a significant challenge. One reason is the apparent contradictory effect that perturbations of some proteins can have on the overall lamellipodium dynamics, depending on exact conditions. Theoretical modelling offers one approach for understanding the balance between the mechanisms that drive and regulate actin network growth and decay. Most models use a 'bottom-up' approach, involving explicitly assembling biochemical components to simulate observable behaviour. Their correctness therefore relies on both the accurate characterization of all the components and the completeness of the relevant processes involved. To avoid potential pitfalls due to this uncertainty, we used an alternative 'top-down' approach, in which measurable features of lamellipodium behaviour, here observed in two different cell types (HL60 and B16-F1), directly inform the development of a simple phenomenological model of lamellipodium dynamics. We show that the kinetics of F-actin association and dissociation scales with the local F-actin density, with no explicit location dependence. This justifies the use of a simplified kinetic model of lamellipodium dynamics that yields predictions testable by pharmacological or genetic intervention. A length-scale parameter (the lamellipodium width) emerges from this analysis as an experimentally accessible probe of network regulatory processes.

Cellular control of cortical actin nucleation.

The contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division [1-3], migration [4], and tissue morphogenesis [5]. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells [6] and during division, where its misregulation can lead to aneuploidy [7]. Despite its importance, our knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence [8-15]. Here, we used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributed a similar amount of F-actin to the cortex but had very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. Our findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator.

Mechanisms of leading edge protrusion in interstitial migration.

While the molecular and biophysical mechanisms underlying cell protrusion on two-dimensional substrates are well understood, our knowledge of the actin structures driving protrusion in three-dimensional environments is poor, despite relevance to inflammation, development and cancer. Here we report that, during chemotactic migration through microchannels with 5 µm × 5 µm cross-sections, HL60 neutrophil-like cells assemble an actin-rich slab filling the whole channel cross-section at their front. This leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a 'free' network that grows from the free membrane at the cell front. Each network is polymerized by a distinct nucleator and, due to their geometrical arrangement, the networks interact mechanically. On the basis of our experimental data, we propose that, during interstitial migration, medial growth of the adherent network compresses the free network preventing its retrograde movement and enabling new polymerization to be converted into forward protrusion.

Highlighting the role of the hydroxyl position on the alkyl spacer of hydroxypropyl-beta-cyclodextrin for enantioseparation in capillary electrophoresis.

This work investigates the resolving power of 2,3-dihydroxypropyl-beta-CD (2,3-DHP-beta-CD) and 3-hydroxypropyl-beta-CD (3-HP-beta-CD) as chiral mobile phase additives (CMPAs) using CE. The effects of experimental parameters (CD concentration, buffer pH, and buffer concentration) on enantiorecognition were investigated. More than 20 basic chiral drugs were resolved with satisfactory enantioselectivity. Comparison with 2-hydroxypropyl-beta-CD (2-HP-beta-CD) showed that one or both of the two new chiral selectors show enhanced enantiorecognition for several molecules with bulky substitutes, such as Zopiclone and Mianserin, however, 2-HP-beta-CD has higher enantiorecognition for most of the analytes. Further studies on the structures of analytes and CMPAs showed that the OH moiety on the propyl spacer plays an important role in the separation of some chiral drugs.

A novel fluorescence-based method in forensic science for the detection of blood in situ.

Full DNA profiles can be generated from just a few cells; however these profiles can be contaminated from other cell types present at the crime scene. We report here on the development of an immunofluorescent technique to spatially locate human-specific blood in situ and also on the ability of this technique to detect individual leukocytes and the DNA contained within them. Four monoclonal mouse anti-human antibodies were evaluated; anti-glycophorin A to detect erythrocytes and anti-CD45, anti-myeloperoxidase (MPO) and anti-histone H1 to detect the nucleated leukocytes. Each antibody was labeled with either Alexa Fluor 488 or 568 for direct application to blood smears which allowed the simultaneous detection of erythrocytes and leukocytes. Furthermore, because histones are DNA binding proteins, the application of anti-histone H1 allowed the detection of DNA within a blood smear. Importantly it was found that full DNA profiles could be achieved after using this method with similar peak area ratios compared to untreated cells. The fluorescent antibodies were found to be human-specific with the exception of anti-histone H1 due to its conserved sequence. However, used in combination with anti-CD45 or anti-MPO the location of DNA from human-specific leukocytes could be detected. The technique was also tested on older blood stains and was still found to be sensitive and cell-specific after 4 months. Following the optimization of the methodology, the fluorescent antibodies were applied to short lengths of black cotton fibres covered with human blood spots. Although the background fluorescence from the cotton was found to be high, erythrocytes and even individual leukocytes could easily be detected, indicating that this technique could be used to detect extremely minute amounts of blood. Used in combination with laser capture microdissection (LCM), this method could be used to pick off individual leukocytes for LCN DNA techniques.

A 150 micron id packed column for the separation of soybean proteins by elution gradient mu-HPLC: simultaneous separation of soybean proteins from cereal and milk proteins.

Mu-HPLC has previously been used to increase the resolution and sensitivity of protein separations but never for the analysis of soybean proteins. In this work, soybean proteins were, for the first time, separated using a capillary column with an internal diameter of 150 microm packed with a Genesis C18 stationary phase (4 microm, 300 angstroms) and UV detection. TFA and acetic acid were investigated as ion-pairing reagents in order to optimise water-ACN gradients to achieve this separation. The column showed good selectivity enabling the separation of soybean proteins from other vegetable proteins such as cereal (wheat, rice and corn) and also from milk proteins. The developed method was applied to the detection of soybean proteins in commercial products elaborated with mixtures of vegetable proteins.