Oligomeric Precursors Research Articles

Species-specific IL-1β is an inflammatory sensor of Seneca Valley Virus 3C Protease.

Inflammasomes play pivotal roles in inflammation by processing and promoting the secretion of IL-1β. Caspase-1 is involved in the maturation of IL-1β and IL-18, while human caspase-4 specifically processes IL-18. Recent structural studies of caspase-4 bound to Pro-IL-18 reveal the molecular basis of Pro-IL-18 activation by caspase-4. However, the mechanism of caspase-1 processing of pro-IL-1β and other IL-1β-converting enzymes remains elusive. Here, we observed that swine Pro-IL-1β (sPro-IL-1β) exists as an oligomeric precursor unlike monomeric human Pro-IL-1β (hPro-IL-1β). Interestingly, Seneca Valley Virus (SVV) 3C protease cleaves sPro-IL-1β to produce mature IL-1β, while it cleaves hPro-IL-1β but does not produce mature IL-1β in a specific manner. When the inflammasome is blocked, SVV 3C continues to activate IL-1β through direct cleavage in porcine alveolar macrophages (PAMs). Through molecular modeling and mutagenesis studies, we discovered that the pro-domain of sPro-IL-1β serves as an 'exosite' with its hydrophobic residues docking into a positively charged 3C protease pocket, thereby directing the substrate to the active site. The cleavage of sPro-IL-1β generates a monomeric and active form of IL-1β, initiating the downstream signaling. Thus, these studies provide IL-1β is an inflammatory sensor that directly detects viral protease through an independent pathway operating in parallel with host inflammasomes.

Internalisation of immunoglobulin light chains by cardiomyocytes in AL amyloidosis: what can biopsies tell us?

Background Cardiac involvement in systemic light chain amyloidosis (AL) leads to chronic heart failure and is a major prognosis factor. Severe cellular defects are provoked in cardiac cells by tissue-deposited amyloid fibrils of misfolded free immunoglobulin light chains (LCs) and their prefibrillar oligomeric precursors. Objective Understanding the molecular mechanisms behind cardiac cell cytotoxicity is necessary to progress in therapy and to improve patient management. One key question is how extracellularly deposited molecules exert their toxic action inside cardiac cells. Here we searched for direct evidence of amyloid LC uptake by cardiomyocytes in patient biopsies. Methods We immunolocalized LCs in cardiac biopsies from four AL cardiac amyloidosis patients and analysed histopathological images by high resolution confocal microscopy and 3D image reconstruction. Results We show, for the first time directly in patient tissue, the presence of LCs inside cardiomyocytes, and report their proximity to nuclei and to caveolin-3-rich areas. Our observations point to macropinocytosis as a probable mechanism of LC uptake. Conclusions Internalisation of LCs occurs in patient cardiomyocytes. This event could have important consequences for the pathogenesis of the cardiac disease by enabling interactions between amyloid molecules and cellular organelles inducing specific signalling pathways, and might bring new insight regarding treatment.

Enhanced Inhibition of Amyloid Formation by Heat Shock Protein 90 Immobilized on Nanoparticles.

As the population ages, an epidemic of neurodegenerative diseases with devastating social consequences is looming. To address the pathologies leading to amyloid-related dementia, novel therapeutic strategies must be developed for the treatment or prevention of neural protein-folding disorders. Nanotechnology will be crucial to this scenario, especially in the design of nanoscale systems carrying therapeutic compounds that can navigate the nervous system and identify amyloid to treat it in situ. In this line, we have recently designed a highly simplified and versatile nanorobot consisting of a protein coating based on the heat shock protein 90 (Hsp90) chaperone that not only propels nanoparticles using ATP but also endows them with the extraordinary ability to fold and restore the activity of heat-denatured proteins. Here, we assess the effectiveness of these nanosystems in inhibiting/reducing the aggregation of amyloidogenic proteins. Using Raman spectroscopy, we qualitatively and quantitatively analyze amyloid by identifying and semi-quantifying the Amide I band. Our findings indicate that the coupling of Hsp90 to nanoparticles results in a more potent inhibition of amyloid formation when compared to the soluble protein. We propose that this enhanced performance may be attributed to enhanced release-capture cycles of amyloid precursor oligomers by Hsp90 molecules nearby on the nanosurface. Intelligent biocompatible coatings, like the one described here, that enhance the diffusivity and self-propulsion of nanoparticles while enabling them to carry out critical functions such as environmental scanning, identification, and amyloid prevention, present an exceptional opportunity for the development of advanced nanodevices in biomedical applications. This approach, which combined active biomolecules with synthetic materials, is poised to reveal remarkable prospects in the field of nanomedicine and biotechnology.

Liquid-Liquid Phase Separation of Intrinsically Disordered Proteins: A New Phase in Our Laboratory

Our laboratory at the Indian Institute of Science Education and Research (IISER) Mohali is involved in studying the intriguing conformational. characteristics of intrinsically disordered proteins (IDPs) that do not fold up into a well-defined 3D structure and exist as rapidly fluctuating conformational ensemble. Our research over the past decade or so has dealt with the IDPs and partially unfolded proteins and their conversion into amyloid aggregates that are associated with deadly neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. We were particularly interested in characterizing the monomeric and oligomeric precursors of amyloid fibrils. This work has been summarized in an invited Feature Article. The current article primarily deals with the newer research direction of our lab in liquid- liquid phase separation of IDPs. Here we summarize our recent exciting discoveries in phase separation of range of disease-related IDPs. Below we provide a background of the emerging field of biomolecular condensates and describe our published results.

Blocking huntingtin oligomers from binding lipid membranes improves phenotype in a C. elegans model of Huntington's disease.

As protein aggregation is the defining hallmark of all amyloid diseases, a common therapeutic strategy is to develop molecules that inhibit aggregation. However, this approach has yielded limited success. Many amyloid proteins directly interact with lipid membranes. These interactions promote distinct aggregation pathways and often result in membrane damage leading to toxicity. As a result, directly targeting the ability of amyloids to bind lipid membranes represents a novel therapeutic strategy. As a proof of principle, the interaction between lipid membranes and mutant huntingtin (htt) aggregates was used to test this strategy. Using a colorimetric lipid binding assay over 1200 compounds were screened for their ability to block htt/lipid binding. The screen was set up to only identify compounds that directly interacted with htt, not the lipid membrane. Two promising compounds Ro-90-7501 and Benzamil hydrochloride were identified. Validation was achieved via two additional methods, a calcein dye leakage assay and in situ atomic force microscopy (AFM). As these compounds directly interact with htt, ThT and AFM assays were performed to assess their impact on aggregation. Ro-90-7501 had a slight inhibitory effect on fibril formation, but aggregate morphologies were unchanged. Benzamil did not inhibit fibril formation; however, oligomer precursors were significantly smaller when exposed to Benzamil. Molecular dynamic simulations (MD) revealed that the two compounds have unique mechanisms of interaction with htt aggregates. Having established that the compound prevented htt from binding membranes, a C. elegans model of Huntington's disease was used to determine if this strategy could alleviate phenotype. Despite have a minimal impact on punctate formation, both compounds reduced a thrashing deficit in animals caused by mutant htt expression, suggesting that this strategy reduces htt toxicity.

Perturbing the conformation of diabetes-associated oligomers using molecular foldamers.

The islet amyloid polypeptide (IAPP) is a 37 aa peptide hormone secreted from pancreatic β-cells. In patients with Type II Diabetes, IAPP undergoes a toxic gain-of-function that results in the formation of amyloid fibrils. While these fibrils are a hallmark of the disease, soluble oligomeric precursors are most potently linked with IAPP pathogenicity. IAPP oligomers (IAos) disrupt homeostasis through a variety of mechanisms, including mitochondrial stress, aberrant Ca2+ signaling, and the formation of membrane pores. However, the biophysics of IAo pathogenicity is poorly characterized due to their instability in solution. We have shown that sodium dodecyl sulfate (SDS) micelles (which mimic anionic membranes) can stabilize IAos of different sizes in a physiological buffer. However, the morphology and conformational diversity of IAPP at the lipid interface is still unclear. We hypothesize that IAo first assembles on membrane surfaces and forms oligomers, which enables misfolding and their toxic gain-of-function. To gain molecular insight into the formation of IAo at this interface, we employ replica-exchange molecular dynamics (MD) and classical MD simulations. We monitor the conformation of IAo in the presence of various protein/micelle ratios as well as in free detergent. Finally, we introduce a molecular foldamer—shown to rescue IAPP-induced toxicity in cell models—to understand its effect on its conformational landscape. By comparing accessible conformations with and without the foldamer, we hope to identify conformations stabilized by the binding interaction. Molecular foldamers demonstrate the therapeutic potency of conformational modulators towards treating IAPP toxicity in T2D. Furthermore, this general strategy can be applied to other disordered peptides (e.g. amyloid-β) that undergo pathological misfolding.

Continuous Flow Microfluidic Production of Arbitrarily Long Tubular Liquid Crystal Elastomer Peristaltic Pump Actuators.

While liquid crystal elastomers (LCEs) are ideal materials for soft-robotic actuators, filling the role of muscle and shape-defining material simultaneously, it is non-trivial to give them ground state shapes beyond simple sheets or fibers. Here tubular LCE actuators scalable to arbitrary length are producedusing a continuous three-phase coaxial flow microfluidic process. By pumping an oligomeric precursor solution between inner and outer aqueous phases in a cylindrically symmetric nested capillary set-up, and by reducing the interfacial tension to negligible values using surfactants adapted to each phase, the tubular liquid flow is stabilized over distances more than 200 times the diameter or 2000 times the thickness. In situ photocrosslinking of the middle phase turns it into an LCE network that is flow-aligned by the shear gradient over the phase. The reversible actuation of the tubes upon heating yields a reduction of the interior space, pumping out enclosed fluid, and the relaxation upon cooling leads to the fluid being sucked back in. By moving a local heat source along the tube, it acts as a peristaltic pump. It is proposed that the tubes could, pending functionalization for light-triggered actuation, function as active synthetic vasculature in biologicalcontexts.

Pure aliphatic polycarbonate networks via photoinduced anionic ring-opening polymerization at elevated temperature

Aliphatic polycarbonates are highly regarded due to their potential as sustainable, biodegradable and inherently biocompatible materials. Therefore, much effort has been directed towards implementation of aliphatic polycarbonates into network structures. To date, these efforts have relied on incorporation of main- or sidechain functional groups into carbonate-containing monomers or oligomeric precursors, with pure polycarbonate networks remaining inaccessible. Here we show that recently developed thermally stable photobase generators have now made cyclic carbonates available for light-induced anionic ring-opening polymerization for the first time to form pure aliphatic polycarbonate networks at elevated temperatures. To gain insights into the polymerization mechanism, the reactivity of synthesized aromatically and aliphatically substituted cyclic carbonates was investigated utilizing photo-DSC and photorheology between 70 and 120 °C. A bifunctional cyclic carbonate was synthesized as crosslinker and tested for network formation with varying amounts of the monofunctional cyclic carbonate as reactive diluent. Tensile tests and DMTA measurements revealed tough, highly tunable (thermo-)mechanical properties of the obtained materials, depending on the ratio of reactive diluent to crosslinker.

Reactive Processing of Acrylic-Based Thermoplastic Composites: A Mini-Review

The demand for thermoplastic composites is continuously increasing because these materials offer many advantages over their thermoset counterparts, such as high toughness, long storage time, easy repairing and recycling, and ability to be thermoformed and heat-welded. However, the manufacturing of thermoplastic composite parts using liquid composite moulding techniques (e.g. resin transfer moulding, vacuum assisted resin transfer moulding … ) is often tricky in the case of melt processing where high temperature and pressure should be chosen to impregnate the fibre reinforcement because of the high melt viscosity of thermoplastics. These issues may be overcome by means of reactive processing where a fibrous preform is first impregnated by a low viscosity mono- or oligomeric precursor and the polymerization of the thermoplastic matrix then occurs in-situ. This article draws a state of the art on the manufacturing characteristics of continuous fibre reinforced acrylic-based reactive thermoplastics (e.g. polymethymethacrylate (PMMA) such as Elium®), which are becoming more and more popular compared to other fast curing thermosets and thermoplastics for in-situ polymerization. Techniques for the in-situ polymerization of methymethacrylate monomers, characterization and modelling of the rheological properties and polymerization kinetics, and some manufacturing related issues such as polymerization shrinkage are reviewed. Particular features of the use of reactive PMMA in different manufacturing techniques of continuous fibre reinforced composites and potential industrial applications are also introduced. Finally, some perspectives for the academic research and industrial development are proposed.

Observation of Oligomeric States Indicates a High Structural Flexibility Required for the Onset of Polyglutamine Fibrillization.

Polyglutamine (polyQ) diseases are caused by misfolding and aggregation of expanded polyQ tracts in the affected protein. PolyQ fibrils have been studied in detail; however, less is known about oligomeric precursor states. By a combination of time-resolved temperature-jump (T-jump) infrared (IR) spectroscopy and an appropriately tailored polyQ model peptide, we succeeded in disentangling conformational dynamics in the heterogeneous ensemble of states evolving during aggregation. Individual structural elements could be differentiated by IR-specific signatures, i.e., hairpin monomers, β-structured oligomers, and disordered structure. Submillisecond dynamics were observed for early oligomeric states in contrast to the slow dynamics of fibril growth. We propose that a high structural flexibility of oligomers is required to initiate fibril formation, but not after a fibrillar structure has consolidated and the fibril just grows. Our study reveals that structural flexibility changes at different stages in the aggregation process, from fibril initiation to fibril growth.

Enhancing the Intermolecular Interactions of Ladder-Type Heteroheptacene-Based Nonfullerene Acceptors for Efficient Polymer Solar Cells by Incorporating Asymmetric Side Chains

Enhancing the Intermolecular Interactions of Ladder-Type Heteroheptacene-Based Nonfullerene Acceptors for Efficient Polymer Solar Cells by Incorporating Asymmetric Side Chains

Effect of alkoxysilane on early age hydration in portland cement pastes

Silanes added during mortar or concrete preparation, may modify the fresh state properties, hydration kinetics and mechanical strength of the final product. The effects of silanes on cement hydration have been widely studied in the literature, however, there is some controversy about its effect at short ages. The present study was undertaken to determine the effect of a TEOS-based alkoxysilane (UCA-T), produced by ultrasound-assisted pre-hydrolysis of an oligomeric precursor, on early age cement paste hydration. The nature of the processes modifying the various stages of cement hydration kinetics in the presence of the alkoxysilane was ascertained by analysing paste composition at several ages (defined on the grounds of calorimetric curve results) using XRD, TG-DTG, FTIR and Raman spectroscopy. The calorimetric curve of pastes containing UCA-T exhibited a new early age, pre-induction period exothermal peak, indicative of UCA-T hydrolysis, C3A and C3S dissolution and ettringite and C–S–H gel precipitation. Portlandite, however, did not precipitate but reacts with the Si(OH)4 sourced from UCA-T hydrolysis to generate further C–S–H gel. The induction period following on that new exothermal peak was considerably longer than the period observed in the reference cement, an effect that intensified at higher UCA-T content.

Single-molecule analysis of soluble IAPP oligomers at diverse membrane surfaces

Islet amyloid polypeptide (IAPP) is a 37 aa peptide that accumulates and fibrilizes in insulin-secreting β-cells in patients with type II diabetes. However, while pancreatic IAPP fibrils are a hallmark of the disease, their soluble oligomeric precursors are most potently correlated with their pathological function. IAPP oligomers (IAo) are thought to disrupt homeostasis through interactions with the proteasomal pathway and direct binding to physiological membranes, leading to membrane-poration and mitochondrial leakage.

Diarylpnictogenyldialkylalanes─Synthesis, Structures, Bonding Analysis, and CO2 Capture.

Several new diphenylamino- and diphenylphosphanyldialkylalanes are reported, which were characterized in solution and in the solid state, assisted by in-depth bonding analysis within the DFT framework. In the case of bulky alkyl substituents on the aluminum atom, the species are stable in their monomeric form and were structurally characterized by single crystal X-ray diffraction, expanding the relatively small field of monomeric pnictogenylalanes. In the case of oligomeric diphenylpnictogenyldimethylalanes, their reactivity toward different σ-donor ligands was studied, and several examples of monomeric adducts could be structurally characterized, including the first cyclic(alkyl)(amino)carbene complexes. The reactivity of these CAAC complexes, their oligomeric precursors, and an unstabilized monomeric aminoalane toward CO2 was probed, leading to different insertion products that could be characterized. Additionally, the mechanism was elucidated by DFT calculations.

Dual approach of bimodality and nano-reinforcement towards toughened PDMS based foul release coatings

It is well established that crosslinked polydimethylsiloxane (PDMS) based coatings have efficacious foul release characteristics. However, a trade-off between mechanical integrity of filled and crosslinked PDMS based coatings and their foul release performance has been a bottleneck for producing efficient and durable coatings. In the present study, we report a dual approach of nano-reinforcement of a bimodal PDMS network as a strategy to produce simultaneously reinforced and toughened PDMS networks with facile release of macrofoulants from their surfaces. The nanocomposites of both unimodal and bimodal PDMS networks were prepared using oligomeric dihydroxyl functional PDMS precursor chains differing in their molecular weights by a factor of five and commercial Cloisite-20A nanoclay as the nanofiller. The clay layers were found to be mildly intercalated in the PDMS matrix, as revealed from investigations by scattering and imaging techniques at different length scales. While the unimodal PDMS networks did show nanoclay induced simultaneous reinforcement and toughening, at equivalent clay loadings, the bimodal PDMS nanocomposites seemed to stretch further with characteristic strain hardening before fracture. Dynamic mechanical analysis (DMA) and swelling studies of the nancomposites further confirmed the nanoclay induced reinforcement effect of the bimodal PDMS host matrix. The intrinsic low surface energy characteristics of PDMS were retained by bimodal blending of long and short chains and its subsequent nano-reinforcement. Macrofouling studies by panel immersion and release force measurements revealed that the macrofoulants could be dislodged from the nanocomposite coatings with a shear force < 0.05 MPa.

Design of cross-linked polyisobutylene matrix for efficient encapsulation of quantum dots.

Photoluminescent quantum dots (QDs) are a prominent example of nanomaterials used in practical applications, especially in light-emitting and light-converting devices. Most of the current applications of QDs require formation of thin films or their incorporation in solid matrices. The choice of an appropriate host material capable of preventing QDs from degradation and developing a process of uniform incorporation of QDs in the matrix have become essential scientific and technological challenges. In this work, we developed a method of uniform incorporation of Cu–Zn–In–S (CZIS) QDs into a highly protective cross-linked polyisobutylene (PIB) matrix with high chemical resistance and low gas permeability. Our approach involves the synthesis of a methacrylate-terminated three-arm star-shaped PIB oligomeric precursor capable of quick formation of a robust 3D polymer network upon exposure to UV-light, as well as the design of a special ligand introducing short PIB chains onto the surface of the QDs, thus providing compatibility with the matrix. The obtained cross-linked QDs-in-polymer composites underwent a complex photostability test in air and under vacuum as well as a chemical stability test. These tests found that CZIS QDs in a cross-linked PIB matrix demonstrated excellent photo- and chemical stability when compared to identical QDs in widely used polyacrylate-based matrices. These results make the composites developed excellent materials for the fabrication of robust, stable and durable transparent light conversion layers.

Cationic, Anionic, and Amphoteric Dual pH/Temperature-Responsive Degradable Microgels via Self-Assembly of Functionalized Oligomeric Precursor Polymers

Multiresponsive smart materials with the capacity to reversibly change properties (i.e., size, charge) upon the application of more than one stimulus (i.e., temperature, pH) offer potential in numerous biotechnology and biomedical applications. However, their typical lack of degradability limits their potential in vivo use. Herein, we demonstrate the use of an aqueous thermally driven self-assembly approach based on hydrazide- and aldehyde-functionalized poly(N-isopropylacrylamide) (PNIPAM) oligomers functionalized with pH-ionizable cationic or anionic comonomers for fabricating degradable temperature/pH dual-responsive microgels. The self-assembled microgels show properties analogous to conventional cationic or anionic PNIPAM microgels, retaining their thermal responsiveness while exhibiting pH-driven swelling upon functional comonomer ionization. Amphoteric microgels can also be produced by mixing cationic- and anionic-functionalized precursor polymers during the self-assembly process that reproduce the high-pH/low-pH parabolic swelling response observed in conventional amphoteric microgels. Coupling the precise dual-responsive swelling responses achievable with the degradability of the hydrazone cross-links, self-assembled charged PNIPAM microgels offer potential for improved performance in drug delivery applications requiring dual pH/temperature-specific delivery (e.g. infection sites or cancer).

Multiturn Hollow Helices: Synthesis and Folding of Long Aromatic Oligoamides.

Aromatic oligoamides adopting helical conformations are synthesized by coupling carboxyl-terminated basic units having two, four, and eight residues to amine-terminated oligomer precursors. Coupling yields show no noticeable reduction with the size of the basic units or the final product. One- and two-dimensional NMR spectroscopy and computational studies demonstrate the reliable helical folding of these oligomers. The X-ray structure of 16mer 7 reveals a compact, multiturn helix having a 9 Å inner pore.

Increasing the Functional Group Diversity in Helical β-Peptoids: Achievement of Solvent- and pH-Dependent Folding.

We report the synthesis of a series of bis-functionalized β-peptoid oligomers of the hexamer length. This was achieved by synthesizing and incorporating protected amino- or azido-functionalized chiral building blocks into precursor oligomers by a trimer segment coupling strategy. The resulting hexamers were readily elaborated to provide target compounds displaying amino groups, carboxy groups, hydroxy groups, or triazolo-pyridines, which should enable metal ion binding. Analysis of the novel hexamers by circular dichroism (CD) spectroscopy and 1H-13C heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy revealed robust helical folding propensity in acetonitrile. CD analysis showed a solvent-dependent degree of helical content in the structural ensembles when adding different ratios of protic solvents including an aqueous buffer. These studies were enabled by a substantial increase in solubility compared to previously analyzed β-peptoid oligomers. This also allowed for the investigation of the effect of pH on the folding propensity of the amino- and carboxy-functionalized oligomers, respectively. Interestingly, we could show a reversible effect of sequentially adding acid and base, resulting in a switching between compositions of folded ensembles with varying helical content. We envision that the present discoveries can form the basis for the development of functional peptidomimetic materials responsive to external stimuli.

Vat photopolymerization of liquid, biodegradable PLGA-based oligomers as tissue scaffolds

Additive manufacturing (AM) of tissue scaffolds provides medical professionals and patients with personalized treatment options, which align with patients’ specific physiology and provide a more efficient and precise manufacturing process. Vat photopolymerization (VP) AM affords biocompatible, degradable, and high-resolution scaffolds capable of achieving specific pore sizes necessary for tissue regeneration. Earlier biodegradable, photoactive polymers for the VP platform usually require dissolution in a solvent or water, which often results in anisotropic shrinkage upon drying and inconsistencies in the final part geometry. This work describes a photoactive oligomeric precursor that eliminates the requirement for water or solvent during VP. Poly(lactide-co-glycolide) (PLGA) diacrylates with low number-average molecular weights (< 1400 g mol−1) provided liquid, viscous precursors. Upon crosslinking, the networks exhibited mechanical strength and low cytotoxicity, which are prerequisites for soft tissue scaffolds. Systematically varying the ratios of lactide to glycolide provided a range of degradation rates from 4 to 12 wks. Tailored degradation rates will enable use in different anatomical environments. Cytotoxicity and cell attachment studies revealed crosslinked films that promoted cell growth and proliferation. This manuscript describes an expanded polymer toolbox for AM of tissue scaffolds with precise resolution in the absence of solvents.