Fibril-like Structures Research Articles

Investigating the deposition of fibrous zeolite particles on leaf surfaces: A novel low-cost method for detecting the presence of airborne hazardous mineral fibers

Naturally occurring fibrous minerals, such as erionite, can pose a significant threat to human health when disturbed and subsequently respired. Understanding the spatial abundance and characteristics of these hazardous fibrous minerals in ambient air is crucial for minimizing human exposure and assessing risk. Conventional detection methods for airborne hazardous mineral fibers, such as those developed for asbestos, are of limited utility in environmental settings where fiber concentrations are low and different fiber types may be present and can be costly especially when monitoring large areas over long periods of time. This study presents an innovative methodology for detecting and identifying the presence of airborne naturally occurring fibrous zeolites, using leaf surface deposition sampling, SEM-EDX analysis for the detection and assessment of elemental composition, and TEM-SAED with continuous rotation diffraction (MicroED) to determine their crystallographic unit cell parameters. In total, 309 fibrous zeolite particles (FZPs) were identified on a range of tree leaf surfaces across 80% of the sampling sites located close to both active and disused zeolite quarries in the Taupo Volcanic Region, New Zealand. The FZPs displayed various morphologies including aggregates, bundles, and fibril-like structures. Of the FZPs detected, 91.2% were < 5µm in length. Tetrahedral Si:(Si+Al) ratio results indicated that 40% of the FZPs were in the reference range for zeolite mordenite. TEM-SAED plus MicroED analysis resulted in 61% of tested FZPs indexed to unit cell parameters that matched with mordenite. This research demonstrates the potential of leaf sampling as a cost-effective method for detecting airborne FZPs while the MicroED data can be utilized for distinguishing between different types of airborne fibrous zeolites in ambient air.

Design of parallel 𝛽-sheet nanofibrils using Monte Carlo search, coarse-grained simulations, and experimental testing.

Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel β-sheets", that is, β-sheets with N-terminus-to-C-terminus 𝛽-strand vectors oriented in parallel. To accomplish the desired β-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-β spine defined by Sawaya et al. This molecular configuration includes two layers of parallel β-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent β-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel 𝛽-sheet arrangement, two formed fibril-like structure with <50% in parallel 𝛽-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel β-sheets based on Fourier transform infrared spectroscopy, circular dichroism, electron microscopy, and thioflavin-T fluorescence spectroscopy measurements.

Deformation mechanism of high-density polyethylene/liquid paraffin films during stretching

In this study, the deformation mechanism of high-density polyethylene (HDPE)/liquid paraffin (LP) films is systematically investigated. The findings demonstrates that the structural changes are significantly influenced by the LP content. Pure HDPE displays a pronounced cavitation phenomenon, whereas HDPE/LP films deform without cavitation. The yielding mechanism observed in HDPE/LP films is related with the extension of molecular chains and rotation/shearing of the lamellae. At high strains, pure HDPE and LP content lower than 20 % form a fibril-like crystal structure. However, samples with LP content exceeding 40 % form a highly oriented and periodically arranged fibrillar crystal structure. Last but not least, distinctly different cross-section morphologies are observed after extraction for HDPE/LP films. When LP content is below 20 %, the films exhibit a non-porous morphology. When the LP content exceeds 40 %, a loose porous morphology is observed after extraction. These findings provide valuable insights into the structural evolution and deformation behavior of HDPE/LP films under mechanical stress.

Investigating the binding interaction of quinoline yellow with bovine serum albumin and anti-amyloidogenic behavior of ferulic acid on QY-induced BSA fibrils

Protein aggregation induces profound changes in the structure along with the conformation of the protein, and is responsible for the pathogenesis of a number of neurodegenerative conditions such as Huntington’s, Creutzfeldt–Jacob, Type II diabetes mellitus, Alzheimer’s, etc. Numerous multi-spectroscopic approaches and in-silico experiments were utilized to investigate BSA’s biomolecular interaction and aggregation in the presence of quinoline yellow. The present research investigation evaluated the interaction of BSA with the food colorant (QY) at two different pH (7.4 and 2.0). The development of the BSA-QY complex was established with UV visible and fluorescence spectroscopy. The quenching of fluorescence upon the interaction of BSA with QY revealed the static nature of quenching mechanism. The Kb value obtained from our result is 4. 54 × 10−4 M−1. The results from the competitive site marker study infer that quinoline yellow is binding with the sub-domain IB of bovine serum albumin, specifically on site III. Three-dimensional fluorescence and synchronous fluorescence spectroscopy were applied for monitoring the alterations in the microenvironment of BSA upon the addition of quinoline yellow. The results from turbidity and RLS studies showed that higher concentrations of QY (80–400 µM) triggered bovine serum albumin (BSA) aggregation at pH 2.0. At pH 7.4, QY couldn’t manage to trigger bovine serum albumin aggregation, perhaps because of the repulsion between negatively charged dye (QY) and anionic bovine serum albumin. The results from far-UV CD, Congo Red, and scanning electron microscopy implicate that the QY-induced aggregates exhibit amyloid fibril-like structures. Molecular docking results revealed that hydrophobic interactions, hydrogen bonding, and Pi-Sulfur interactions contribute to QY-induced aggregation of BSA. Further, the amyloid inhibitory potential of ferulic acid (FA), a phenolic acid on QY-induced aggregation of BSA, has also been assessed. The QY-induced amyloid fibrils are FA-soluble, as confirmed by turbidity, RLS, and far-UV CD studies. Far-UV CD results showed that FA retains α helix and inhibits cross β sheet formation when the BSA samples were pre-incubated with increasing concentrations of FA (0–500 µM). Our findings conclude that QY dye successfully stimulates BSA aggregation, but ferulic acid inhibits QY-induced aggregation of BSA. Thus, FA can serve as a therapeutic agent and can help in the treatment of various amyloid-related conditions.

Nucleotide-based regenerated fiber production using salmon (Oncorhynchus keta) milt waste by solution spinning

The use of nucleic acid-derived fibers has not been developed in contrast to the traditional use of polysaccharide- and protein-based fibers in daily life. Salmon, Oncorhynchus keta, is an abundant fishery resource, and its milt contains a huge amount of DNA. Most of the milt is discarded because it degrades easily and is unsuitable for food consumption. DNA-based fibers are expected to possess functionality and mechanical strength because DNA is a polyanion with a high molecular weight. Here, using DNA extracted from the salmon milt, we produced nucleotide-based fibers. A solution spinning system was applied using ethanol as a coagulant. Adding the salt to the dope solution reduced the solubility of DNA, which was essential for the successful spinning of DNA-based fibers. The obtained fibers became insoluble in water by ultraviolet (UV) exposure. Fibril-like structures were detected on the fracture surface, and humidity influenced the conformational structure. This study focuses on the bulk-scale production of biodegradable DNA-based fibers. Therefore, it can be used not only for clothing and filters but also as a functional material to remove harmful pollutants released into the ocean, such as heavy metal ions and aromatic derivatives.

Aβ Amyloid Fibers Drastically Alter the Topography and Mechanical Properties of Lipid Membranes.

Alzheimer's disease (AD) is related to the fibrillation of the Aβ peptides at neuronal membranes, a process that depends on the lipid composition and may impart different physical states to the membrane. In the present work, we study the properties of the Aβ peptide when mixed with a zwitterionic lipid (DMPC), using the Langmuir monolayer technique as an approach to control membrane physical conditions. First, we build on previous characterizations of pure Aβ monolayers and observe that, in addition to high shear, these films present a pronounced compressional hysteresis. When Aβ is assembled with DMPC in a binary film, the resulting membranes become heterogeneous, with a peptide-enriched phase distributed in a network-like pattern, and they exhibit a lateral transition that depends on the Aβ content. At lower peptide proportions, the films segregate into two well-defined phases: one consisting of lipids and another enriched with peptides. The reflectivity of these phases differs from that obtained for pure Aβ films. Thus, the formed fibers effectively cover most of the interface area and remain stable at higher pressures (from 20 to 30 mN m-1 depending on Aβ content) compared to pure peptide films (17 mN m-1). Furthermore, such structures induce a compressional hysteresis in the film, similar to that of pure peptide films (which is nonexistent in the pure lipid monolayer), even at low peptide proportions. We claim that the mechanical properties at the interface are governed by the size of the fibril-like structures. Based on the low molar fractions and surface packing at which these phenomena were observed, we postulate that as a consequence of peptide intermolecular interactions, Aβ may have drastic effects on the molecular arrangement and mechanical properties of a lipid membrane.

Revealing Deformation Mechanisms in Polymer-Grafted Thermoplastic Elastomers via In Situ Small-Angle X-ray Scattering.

The tunable properties of thermoplastic elastomers (TPEs), through polymer chemistry manipulations, enable these technologically critical materials to be employed in a broad range of applications. The need to "dial-in" the mechanical properties and responses of TPEs generally requires the design and synthesis of new macromolecules. In these designs, TPEs with nonlinear macromolecular architectures outperform the mechanical properties of their linear copolymer counterparts, but the differences in the deformation mechanism providing enhanced performance are unknown. Here, in situ small-angle X-ray scattering (SAXS) measurements during uniaxial extension reveal distinct deformation mechanisms between a commercially available linear poly(styrene)-poly(butadiene)-poly(styrene) (SBS) triblock copolymer and the grafted SBS version containing grafted poly(styrene) (PS) chains from the poly(butadiene) (PBD) midblock. The neat SBS (φSBS = 100%) sample deforms congruently with the macroscopic dimensions, with the domain spacing between spheres increasing and decreasing along and transverse to the stretch direction, respectively. At high extensions, end segment pullout from the PS-rich domains is detected, which is indicated by a disordering of SBS. Conversely, the PS-grafted SBS that is 30 vol % SBS and 70% styrene (φSBS = 30%) exhibits a lamellar morphology, and in situ SAXS measurements reveal an unexpected deformation mechanism. During deformation, there are two simultaneous processes: significant lamellar domain rearrangement to preferentially orient the lamellae planes parallel to the stretch direction and crazing. The samples whiten at high strains as expected for crazing, which corresponds with the emergence of features in the 2D SAXS pattern during stretching consistent with fibril-like structures that bridge the voids in crazes. The significant domain rearrangement in the grafted copolymers is attributed to the new junctions formed across multiple PS domains by the grafting of a single chain. The in situ SAXS measurements provide insights into the enhanced mechanical properties of grafted copolymers that arise through improved physical cross-linking that leads to nanostructure domain reorientation for self-reinforcement and craze formation where fibrils help to strengthen the polymer.

Interfacial Aβ fibril formation is modulated by the disorder-order state of the lipids: The concept of the physical environment as amyloid inductor in biomembranes

The behavior of amphiphilic molecules such as lipids, peptides and their mixtures at the air/water interface allow us to evaluate and visualize the arrangement formed in a confined and controlled surface area. We have studied the surface properties of the zwitterionic DPPC lipid and Aβ(1–40) amyloid peptide in mixed films at different temperatures (from 15 to 40 °C). In this range of temperature the surface properties of pure Aβ(1–40) peptide remained unchanged, whereas DPPC undergoes its characteristic liquid-expanded → liquid-condensed bidimensional phase transition that depends on the temperature and lateral pressure. This particular property of DPPC makes it possible to dynamically study the influence of the lipid phase state on amyloid structure formation at the interface in a continuous, isothermal and abrupt change on the environmental condition. As the mixed film is compressed the fibril-like structure of Aβ(1–40) is triggered specifically in the liquid-expanded region, independently of temperature, and it is selectively excluded from the well-visible liquid condensed domains of DPPC. The Aβ amyloid fibers were visualized by using BAM and AFM and they were Thio T positive. In mixed DPPC/Aβ(1–40) films the condensed domains (in between 11 mN/m to 20 mN/m) become irregular probably due to the fibril-like structures is imposing additional lateral stress sequestering lipid molecules in the surrounding liquid-expanded phase to self-organize into amyloids.

Development of chitin nanofiber coatings for prolonging shelf life and inhibiting bacterial growth on fresh cucumbers

The widespread usage of petroleum-based polymers as single-use packaging has had harmful effects on the environment. Herein, we developed sustainable chitin nanofiber (ChNF) coatings that prolong the shelf life of fresh cucumbers and delay the growth of pathogenic bacteria on their surfaces. ChNFs with varying degrees of acetylation were successfully prepared via deacetylation using NaOH with treatment times of 0–480 min and defibrillated using mechanical blending. With longer deacetylation reaction times, more acetamido groups (–NHCOCH3) in chitin molecules were converted to amino groups (–NH2), which imparted antibacterial properties to the ChNFs. The ChNF morphologies were affected by deacetylation reaction time. ChNFs deacetylated for 240 min had an average width of 9.0 nm and lengths of up to several μm, whereas rod-like structured ChNFs with a mean width of 7.3 nm and an average length of 222.3 nm were obtained with the reaction time of 480 min. Furthermore, we demonstrated a standalone ChNF coating to extend the shelf life of cucumbers. In comparison to the rod-like structured ChNFs, the 120 and 240-min deacetylated ChNFs exhibited a fibril-like structure, which considerably retarded the moisture loss of cucumbers and the growth rate of bacteria on their outer surfaces during storage. Cucumbers coated with these 120 and 240-min deacetylated ChNFs demonstrated a lower weight loss rate of ⁓ 3.9% day−1 compared to the uncoated cucumbers, which exhibited a weight loss rate of 4.6% day−1. This protective effect provided by these renewable ChNFs holds promising potential to reduce food waste and the use of petroleum-based packaging materials.

The interwoven fibril-like structure of amyloid-beta plaques in mouse brain tissue visualized using super-resolution STED microscopy

BackgroundStandard neuropathologic analysis of Alzheimer’s brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth.ResultsIn our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5–10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM.ConclusionsThe utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aβ) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer’s disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer’s research.

Self-Assembled Amino Acid Microstructures as Biocompatible Physically Unclonable Functions (BPUFs) for Authentication of Therapeutically Relevant Hydrogels.

Counterfeited biomedical products result in significant economic losses and pose a public health hazard for over a million people yearly. Hydrogels, a class of biomedical products, are being investigated as alternatives to conventional biomedical products and are equally susceptible to counterfeiting. Here, a biocompatible, physically unclonable function (BPUF) to verify the authenticity of therapeutically relevant hydrogels are developed. The principle of BPUF relies on the self-assembly of tyrosine into fibril-like structures which are incorporated into therapeutically relevant hydrogels resulting in their random dispersion. This unclonable arrangement leads to distinctive optical micrographs captured using an optical microscope. These optical micrographs are transformed into a unique security code through cryptographic techniques which are then used to authenticate the hydrogel. The temporal stability of the BPUFs are demonstrated and additionally, exploit the dissolution propensity of the structures upon exposure to an adulterant to identify the tampering of the hydrogel. Finally, a platform to demonstrate the translational potential of this technology in validating and detecting tampering of therapeutically relevant hydrogels is developed. The potential of BPUFs to combat hydrogel counterfeiting is exemplified by its simplicity in production, ease of use, biocompatibility, and cost-effectiveness.

Temperature diagnostics of chromospheric fibrils

Context. Chromospheric fibrils are thin and elongated structures that connect nearby photospheric magnetic field concentrations of opposite polarities. Aims. We assess the possibilities and drawbacks related to the use of current instrumentation and inversion techniques to infer the thermodynamic structure of chromospheric fibrils. Methods. We employed spectroscopic observations obtained in the Ca II 854.2 nm line with the CRISP instrument at the Swedish 1-m Solar Telescope and in coordination with observations in the ultraviolet Mg II h &amp; k lines taken with the IRIS satellite. We studied the temperature sensitivity of these chromospheric lines to properly invert their spectral profiles with the Stockholm inversion Code and determine the temperature, line-of-sight velocity, and microturbulent velocity of manually traced chromospheric fibrils present in the field of view. Results. Fibril-like structures show a very particular dependence of their temperature as a function of the position along their length. Their temperatures at the detected footpoints are, on average, 300 K higher than the temperature at the midpoint. The temperature variation appears to be almost symmetrical in shape, with partially traced fibrils showing a similar trend for the temperature variation. Additionally, the response of the Ca II 854.2 nm line core to variations of the temperature for the inverted models of the atmosphere in fibril areas seems to be insufficient to properly resolve the aforementioned temperature structure. Only the addition of more temperature sensitive lines such as the Mg II h &amp; k lines would make it possible to properly infer the thermodynamic properties of chromospheric fibrils. Comparisons between the results obtained here and in previous studies focused on bright Ca II K fibrils yield great similarities between these structures in terms of their temperature.

Atomic insights into amyloid-induced membrane damage.

Amphipathic peptides can cause biological membranes to leak either by dissolving their lipid content via a detergent-like mechanism or by forming pores on the membrane surface. These modes of membrane damage have been related to the toxicity of amyloid peptides and to the activity of antimicrobial peptides. Here, we will discuss all-atom simulations in which membrane-bound amphipathic peptides self-assemble into beta-sheets that subsequently either form stable pores inside the bilayer or drag lipids out of the membrane surface. An analysis of these simulations shows that the acyl tail of lipids interact strongly with non-polar side chains of peptides deposited on the membrane. These strong interactions enable lipids to be dragged out of the bilayer by oligomeric structures accounting for detergent-like damage. They also disturb the orientation of lipid tails in the vicinity of peptides. These distortions are minimized around pore structures. We also show that membrane-bound beta-sheets become twisted with one of their extremities partially penetrating the lipid bilayer. This allows peptides on opposite leaflets to interact and form a long transmembrane beta-sheet, which initiates poration. In simulations, where peptides are deposited on a single leaflet, the twist in beta-sheets allows them to penetrate the membrane and form pores. In addition, our simulations show that fibril-like structures produce little damage to lipid membranes, as non-polar side chains in these structures are unavailable to interact with the acyl tail of lipids.

Vascular smooth muscle cell senescence accelerates medin aggregation via small extracellular vesicle secretion and extracellular matrix reorganization.

Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid peptide. Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid accumulation in the extracellular matrix (ECM). To determine the mechanisms of AMA formation with age, we explored the impact of vascular smooth muscle cell (VSMC) senescence, EV secretion, and ECM remodeling on medin accumulation. Medin was detected in EVs secreted from primary VSMCs. Small, round medin aggregates colocalized with EV markers in decellularized ECM in vitro and medin was shown on the surface of EVs deposited in the ECM. Decreasing EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age and VSMC senescence increased EV secretion, increased EV medin loading and triggered deposition of fibril-like medin. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition, which led to enhanced EV-ECM binding and accelerated medin aggregation. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalized with EVs and medin. Isolated EVs selectively bound to HSPG2 in the ECM and its knock-down decreased formation of fibril-like medin structures. These data identify VSMC-derived EVs and HSPG2 in the ECM as key mediators of medin accumulation, contributing to age-associated AMA development.

Melittin-solid phospholipid mixed films trigger amyloid-like nano-fibril arrangements at air-water interface

We used the Langmuir monolayers technique to study the surface properties of melittin toxin mixed with either liquid-condensed DSPC or liquid-expanded POPC phospholipids. Pure melittin peptide forms stable insoluble monolayers at the air-water interface without interacting with Thioflavin T (Th-T), a sensitive probe to detect protein amyloid formation. When melittin peptide is mixed with DSPC lipid at 50 % of peptide area proportion at the surface, we observed the formation of fibril-like structures detected by Brewster angle microscopy (BAM), but they were not observable with POPC. The nano-structures in the melittin-DSPC mixtures became Th-T positive labeling when the arrangement was observed with fluorescence microscopy. In this condition, Th-T undergoes an unexpected shift in the typical emission wavelength of this amyloid marker when a 2D fluorescence analysis is conducted.Even when reflectivity analysis of BAM imaging evidenced that these structures would correspond to the DSPC lipid component of the mixture, the interpretation of ATR-FTIR and Th-T data suggested that both components were involved in a new lipid-peptide rearrangement. These nano-fibril arrangements were also evidenced by scanning electron and atomic force microscopy when the films were transferred to a mica support. The fibril formation was not detected when melittin was mixed with the liquid-expanded POPC lipid. We postulated that DSPC lipids can dynamically trigger the process of amyloid-like nano-arrangement formation at the interface. This process is favored by the relative peptide content, the quality of the interfacial environment, and the physical state of the lipid at the surface.

Atomic Insights into Amyloid-Induced Membrane Damage.

Amphipathic peptides can cause biological membranes to leak either by dissolving their lipid content via a detergent-like mechanism or by forming pores on the membrane surface. These modes of membrane damage have been related to the toxicity of amyloid peptides and to the activity of antimicrobial peptides. Here, we perform the first all-atom simulations in which membrane-bound amphipathic peptides self-assemble into β-sheets that subsequently either form stable pores inside the bilayer or drag lipids out of the membrane surface. An analysis of these simulations shows that the acyl tail of lipids interact strongly with non-polar side chains of peptides deposited on the membrane. These strong interactions enable lipids to be dragged out of the bilayer by oligomeric structures accounting for detergent-like damage. They also disturb the orientation of lipid tails in the vicinity of peptides. These distortions are minimized around pore structures. We also show that membrane-bound β-sheets become twisted with one of their extremities partially penetrating the lipid bilayer. This allows peptides on opposite leaflets to interact and form a long transmembrane β-sheet, which initiates poration. In simulations, where peptides are deposited on a single leaflet, the twist in β-sheets allows them to penetrate the membrane and form pores. In addition, our simulations show that fibril-like structures produce little damage to lipid membranes, as non-polar side chains in these structures are unavailable to interact with the acyl tail of lipids.

Truncated Tau caused by intron retention is enriched in Alzheimer’s disease cortex and exhibits altered biochemical properties

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β plaques and Tau tangles in brain tissues. Recent studies indicate that aberrant splicing and increased level of intron retention is linked to AD pathogenesis. Bioinformatic analysis revealed increased retention of intron 11 at the Tau gene in AD female dorsal lateral prefrontal cortex as compared to healthy controls, an observation validated by quantitative polymerase chain reaction using different brain tissues. Retention of intron 11 introduces a premature stop codon, resulting in the production of truncated Tau11i protein. Probing with customized antibodies designed against amino acids encoded by intron 11 showed that Tau11i protein is more enriched in AD hippocampus, amygdala, parietal, temporal, and frontal lobe than in healthy controls. This indicates that Tau messenger RNA with the retained intron is translated invivo instead of being subjected to nonsense-mediated decay. Compared to full-length Tau441 isoform, ectopically expressed Tau11i forms higher molecular weight species, is enriched in Sarkosyl-insoluble fraction, and exhibits greater protein stability in cycloheximide assay. Stably expressed Tau11i also shows weaker colocalization with α-tubulin of microtubule network in human mature cortical neurons as compared to Tau441. Endogenous Tau11i is enriched in Sarkosyl-insoluble fraction in AD hippocampus and forms aggregates that colocalize weakly with Tau4R fibril-like structure in AD temporal lobe. The elevated level of Tau11i protein in AD brain tissues tested, coupled with biochemical properties resembling pathological Tau species suggest that retention of intron 11 of Tau gene might be an early biomarker of AD pathology.

Biochemical characterization of medaka (Oryzias latipes) fibrinogen gamma and its gene disruption resulting in anemia as a model fish

At the final stages of blood coagulation, fibrinogen is processed into insoluble fibrin by thrombin resulting in fibril-like structure formation. Via further cross-linking reactions between the fibrin gamma subunit by the catalytic action of blood transglutaminase (Factor XIII), this molecule gains further physical stability. Meanwhile, since fibrinogen is expressed in various cells and tissues, this molecule can exhibit other functions apart from its role in blood coagulation. To create a system studying on aberrant coagulation and investigate the physiological functions, using a model fish medaka (Oryzias latipes), we established gene-deficient mutants of fibrinogen gamma subunit protein in parallel with its biochemical analysis, such as tissue distribution pattern and substrate properties. By genetic deletion via genome editing, two distinct mutants displayed retardation of blood coagulation. The mutants showed lower hematocrit with aberrant erythrocyte maturation, which indicates that fibrin deficiency caused severe anemia, and also appeared as a model for investigation of the fibrin function.

Substoichiometric Inhibition of Insulin against IAPP Aggregation Is Attenuated by the Incompletely Processed N-Terminus of proIAPP.

Substoichiometric aggregation inhibition of human islet amyloid polypeptide (IAPP), the hallmark of type 2 diabetes impacting millions of people, is crucial for developing clinic therapies, yet it remains challenging given that many candidate inhibitors require high doses. Intriguingly, insulin, the key regulatory polypeptide on blood glucose levels that are cosynthesized, costored, and cosecreted with IAPP by pancreatic β cells, has been identified as a potent inhibitor that can suppress IAPP amyloid aggregation at substoichiometric concentrations. Here, we computationally investigated the molecular mechanisms of the substoichiometric inhibition of insulin against the aggregation of IAPP and the incompletely processed IAPP (proIAPP) using discrete molecular dynamics simulations. Our results suggest that the amyloid aggregations of both IAPP and proIAPP might be disrupted by insulin through its binding with the shared amyloidogenic core sequences. However, the N-terminus of proIAPP competed with the amyloidogenic core sequences for the insulin interactions, resulting in attenuated inhibition by insulin. Moreover, insulin preferred to bind the elongation surfaces of IAPP seeds with fibril-like structure, with a stronger affinity than that of IAPP monomers. The capping of elongation surfaces by a small amount of insulin sterically prohibited the seed growth via monomer addition, achieving the substoichiometric inhibition. Together, our computational results provided molecular insights for the substoichiometric inhibition of insulin against IAPP aggregation, also the weakened effect on proIAPP. The uncovered substoichiometric inhibition by capping the elongation of amyloid seeds or fibrils may guide the rational designs of new potent inhibitors effective at low doses.

Structural characterization of fibrils formed by the low complexity domain of the RNA-binding protein TIA-1

Proteins containing low complexity sequence domains harbor mutations genetically linked to neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). Not much is known about the structures these protein domains adopt functionally and what role the mutations play in causing disease. One key protein with mutations present in the low complexity domain (LCD) is T-cell restricted intracellular antigen-1 (TIA1), which forms fibril-like structures that are usually associated with disease.