Binding structures of SERF1a with NT17-polyQ peptides of huntingtin exon 1 revealed by SEC-SWAXS, NMR and molecular simulation.

The aberrant fibrillization of huntingtin exon 1 (Httex1) characterized by an expanded polyglutamine (polyQ) tract is a defining feature of Huntington's disease, a neurodegenerative disorder. Recent investigations underscore the involvement of a small EDRK-rich factor 1a (SERF1a) in promoting Httex1 fibrillization through interactions with its N terminus. By establishing an integrated approach with size-exclusion-column-based small- and wide-angle X-ray scattering (SEC-SWAXS), NMR, and molecular simulations using Rosetta, the analysis here reveals a tight binding of two NT17 fragments of Httex1 (comprising the initial 17 amino acids at the N terminus) to the N-terminal region of SERF1a. In contrast, examination of the complex structure of SERF1a with a coiled NT17-polyQ peptide (33 amino acids in total) indicates sparse contacts of the NT17 and polyQ segments with the N-terminal side of SERF1a. Furthermore, the integrated SEC-SWAXS and molecular-simulation analysis suggests that the coiled NT17 segment can transform into a helical conformation when associated with a polyQ segment exhibiting high helical content. Intriguingly, NT17-polyQ peptides with enhanced secondary structures display diminished interactions with SERF1a. This insight into the conformation-dependent binding of NT17 provides clues to a catalytic association mechanism underlying SERF1a's facilitation of Httext1 fibrillization.

Nanoarchitectonics of Nanocellulose Filament Electrodes by Femtosecond Pulse Laser Deposition of ZnO and In Situ Conjugation of Conductive Polymers.

Electroactive filament electrodes were synthesized by wet-spinning of cellulose nanofibrils (CNF) followed by femtosecond pulse laser deposition of ZnO (CNF@ZnO). A layer of conducting conjugated polymers was further adsorbed by in situ polymerization of either pyrrole or aniline, yielding systems optimized for electron conduction. The resultant hybrid filaments were thoroughly characterized by imaging, spectroscopy, electrochemical impedance, and small- and wide-angle X-ray scattering. For the filaments using polyaniline, the measured conductivity was a result of the synergy between the inorganic and organic layers, while the contribution was additive in the case of the systems containing polypyrrole. This observation is rationalized by the occurrence of charge transfer between ZnO and polyaniline but not that with polypyrrole. The introduced conductive hybrid filaments displayed a performance that competes with that of metallic counterparts, offering great promise for next-generation filament electrodes based on renewable nanocellulose.

Sulfated disaccharide protects membrane and DNA damages from arginine-rich dipeptide repeats in ALS.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most prevalent mutation among amyotrophic lateral sclerosis (ALS) patients. The patients carry over ~30 to hundreds or thousands of repeats translated to dipeptide repeats (DPRs) where poly-glycine-arginine (GR) and poly-proline-arginine (PR) are most toxic. The structure-function relationship is still unknown. Here, we examined the minimal neurotoxic repeat number of poly-GR and found that extension of the repeat number led to a loose helical structure disrupting plasma and nuclear membrane. Poly-GR/PR bound to nucleotides and interfered with transcription. We screened and identified a sulfated disaccharide that bound to poly-GR/PR and rescued poly-GR/PR-induced toxicity in neuroblastoma and C9-ALS-iPSC-derived motor neurons. The compound rescued the shortened life span and defective locomotion in poly-GR/PR expressing Drosophila model and improved motor behavior in poly-GR-injected mouse model. Overall, our results reveal structural and toxicity mechanisms for poly-GR/PR and facilitate therapeutic development for C9-ALS.

Comprehensive characterization of polyproline tri-helix macrocyclic nanoscaffolds for predictive ligand positioning.

Multivalent ligands hold promise for enhancing avidity and selectivity to simultaneously target multimeric proteins, as well as potentially modulating receptor signaling in pharmaceutical applications. Essential for these manipulations are nanosized scaffolds that precisely control ligand display patterns, which can be achieved by using polyproline oligo-helix macrocyclic nanoscaffolds via selective binding to protein oligomers and cell surface receptors. This work focuses on synthesis and structural characterization of different-sized polyproline tri-helix macrocyclic (PP3M) scaffolds. Through combined analysis of circular dichroism (CD), small- and wide-angle X-ray scattering (SWAXS), electron spin resonance (ESR) spectroscopy, and molecular modeling, a non-coplanar tri-helix loop structure with partially crossover helix ends is elucidated. This structural model aligns well with scanning tunneling microscopy (STM) imaging. The present work enhances the precision of nanoscale organic synthesis, offering prospects for controlled ligand positioning on scaffolds. This advancement paves the way for further applications in nanomedicine through selective protein interaction, manipulation of cell surface receptor functions, and developments of more complex polyproline-based nanostructures.

Performance and Solution Structures of Side-Chain-Bridged Oligo (Ethylene Glycol) Polymer Photocatalysts for Enhanced Hydrogen Evolution under Natural Light Illumination.

Converting solar energy into hydrogen energy using conjugated polymers (CP) is a promising solution to the energy crisis. Improving water solubility plays one of the critical factors in enhancing the hydrogen evolution rate (HER) of CP photocatalysts. In this study, a novel concept of incorporating hydrophilic side chains to connect the backbones of CPs to improve their HER is proposed. This concept is realized through the polymerization of carbazole units bridged with octane, ethylene glycol, and penta-(ethylene glycol) to form three new side-chain-braided (SCB) CPs: PCz2S-OCt, PCz2S-EG, and PCz2S-PEG. Verified through transient absorption spectra, the enhanced capability of PCz2S-PEG for ultrafast electron transfer and reduced recombination effects has been demonstrated. Small- and wide-angle X-ray scattering (SAXS/WAXS) analyses reveal that these three SCB-CPs form cross-linking networks with different mass fractal dimensions (f) in aqueous solution. With the lowest f value of 2.64 and improved water/polymer interfaces, PCz2S-PEG demonstrates the best HER, reaching up to 126.9 µmol h-1 in pure water-based photocatalytic solution. Moreover, PCz2S-PEG exhibits comparable performance in seawater-based photocatalytic solution under natural sunlight. In situ SAXS analysis further reveals nucleation-dominated generation of hydrogen nanoclusters with a size of ≈1.5 nm in the HER of PCz2S-PEG under light illumination.

Amyloid modifier SERF1a interacts with polyQ-expanded huntingtin-exon 1 via helical interactions and exacerbates polyQ-induced toxicity.

Abnormal polyglutamine (polyQ) expansion and fibrillization occur in Huntington's disease (HD). Amyloid modifier SERF enhances amyloid formation, but the underlying mechanism is not revealed. Here, the fibrillization and toxicity effect of SERF1a on Htt-exon1 are examined. SERF1a enhances the fibrillization of and interacts with mutant thioredoxin (Trx)-fused Httex1. NMR studies with Htt peptides show that TrxHttex1-39Q interacts with the helical regions in SERF1a and SERF1a preferentially interacts with the N-terminal 17 residues of Htt. Time-course analysis shows that SERF1a induces mutant TrxHttex1 to a single conformation enriched of ß-sheet. Co-expression of SERF1a and Httex1-polyQ in neuroblastoma and lentiviral infection of SERF1a in HD-induced polypotent stem cell (iPSC)-derived neurons demonstrates the detrimental effect of SERF1a in HD. Higher level of SERF1a transcript or protein is detected in HD iPSC, transgenic mice, and HD plasma. Overall, this study provides molecular mechanism for SERF1a and mutant Httex1 to facilitate therapeutic development for HD.

Thermal-/pH-triggered hollow mesoporous carbon nanocarrier for NIR-responsive drug release.

Intelligent drug-delivery systems are considered one of the most important techniques for improving cancer treatment using existing over-the-counter medicines. However, metallic materials are always accompanied by metabolism problems, whereas chemotherapy produces several side effects in humans. Carbon-based materials exhibit exceptional features such as bio-affinity and bio-degradability. Herein, hollow mesoporous carbon nanoparticles (HMCs) are reported as effective nanocarriers of anti-cancer small drug molecules. Near IR (NIR) sources, which can penetrate most organs, induce thermal effects via non-invasive pathways. NIR radiation not only provides thermal therapy but also is compatible with temperature-sensitive coated responsive polymer shells. The template method was used to synthesize HMCs with size 200 ± 50 nm, under various conditions, to obtain suitably sized and hollow structures for liver-cancer treatment. Additional pH/thermal-bi-responsive poly(N-isopropylacrylamide) (PNIPAM) shells were further coated onto the HMCs to produce multiple shells that could trigger swelling motions in PNIPAM@HMCs, as confirmed via small-angle X-ray scattering (SAXS). NIR results demonstrated an extreme increase to the ∆T of 8.7 and 14.2 °C for HMC and PNIPAM@HMCs, respectively. The SAXS spectra analyzed using SasView simulations demonstrated the multi-shell structures of synthesized HMCs and the release mechanism of PNIPAM@HMCs. Based on the model simulation of SAXS, the different rates of polymer swelling indicated the core shrinkage (229.7 to 134.2 Å) and shell expansion (324.3 to 514.3 Å) at 37 °C and 42 °C, respectively. In addition, the first-order, Higuchi, Korsmeyer-Peppas, and Weibull mathematical models were used to verify the drug-release kinetics, and the model with the highest R2 value was considered most suitable for further application. This paper presents the first SAXS study on PNIPAM@HMCs release kinetics and related mechanisms. This phenomenon indicates NIR-induced PNIPAM@HMCs as an effective strategy for cancer treatment via doxorubicin release.

Clay nanosheets simultaneously intercalated and stabilized by PEGylated chitosan as drug delivery vehicles for cancer chemotherapy.

Montmorillonite (MMT) has been frequently utilized as drug vehicles due to its high specific surface area, excellent cation exchange capacity and biocompatibility. However, the significant flocculation of MMT under physiological condition restricted its application to drug delivery. To conquer this problem, the graft-type PEGylated chitosan (PEG-CS) adducts were synthesized as intercalator to stabilize MMT dispersion. Through electrostatic attraction between the chitosan and MMT, the PEG-CS adducts were adsorbed on MMT surfaces and intercalated into MMT. The resulting PEG-CS/MMT nanosheets possessed PEG-rich surfaces, thus showing outstanding dispersion in serum-containing environment. Moreover, the physicochemical characterization revealed that the increased mass ratio of PEG-CS to MMT led to the microstructure transition of PEG-CS/MMT nanosheets from multilayered to exfoliated structure. Interestingly, the PEG-CS/MMT nanosheets with mass ratio of 8.0 in freeze-dried state exhibited a hierarchical lamellar structure organized by the intercalated MMT bundles and unintercalated PEG-CS domains. Notably, the multilayered PEG-CS/MMT nanosheets showed the capability of loading doxorubicin (DOX) superior to the exfoliated counterparts. Importantly, the DOX@PEG-CS/MMT nanosheets endocytosed by TRAMP-C1 cells liberated the drug progressively within acidic organelles, thereby eliciting cell apoptosis. This work provides a new strategy of achieving the controllable dispersion stability of MMT nanoclays towards application potentials in drug delivery.

De novo synthesis of a MIL-125(Ti) carrier for thermal- and pH-responsive drug release.

Microporous round cake-like (diameter: 900 ± 100 nm) MIL-125(Ti) carrier with a central metal (Ti) exhibiting bio-affinity and possessing a great potential to be used as drug release platform, has been synthesized in the present study. The thermal and pH responsiveness of drug delivery systems (DDS) are the most important parameters for drug release and can be provided through polymer coating techniques. The Pluronic F127 (F127) and chitosan (CH) monomers were inserted into the crystal lattice of MIL-125(Ti) carrier during the de novo synthesis process, which were subsequently loaded with doxorubicin (DOX). The results reveal particle size changes (ranged between 30 and 50 %) from the original size of the MIL-125(Ti) carrier in response to temperature and pH when the carrier reaches acid environment. The drug release profiles have been completed through self-design device, which provides for the real-time release in the DOX amounts via UV-Vis spectra. The kinetics analysis was used to evaluate the R2 values of first order, Higuchi, Korsmeyer-peppas, and Weibull fitting equations, where the Weibull fitting indicated the best R2. An increase by 59.3 % of DOX released under the acid status (pH = 5.4) was observed, indicating that the CH-MIL-125(Ti) carrier is temperature and pH responsive. Moreover, the lattice explosion resulting from the temperature increase in the range of 25-42 °C caused an increase in F127-MIL-125(Ti) by 30.8-38.3 %. The simulated SAXS/WAXS studies for the microstructures of MIL-125(Ti) based DDS at different temperatures after polymer coating (F127-MIL-125(Ti)) provide the possible mechanism of lattice explosion. As such, the responsive Ti-MOF has a highly potential for use in the applications of cancer treatment.

Interdomain Flexibility of Chikungunya Virus nsP2 Helicase-Protease Differentially Influences Viral RNA Replication and Infectivity.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome.

Nanostructured silver dendrites for photon-induced Cysteine dimerization.

Under a controlled adsorption environment, L-cysteine molecules can be chemically adsorbed to the dendritic silver (Ag-D) surface by electrochemical methods with different functional groups. It is verified by surface-enhanced Raman spectroscopy that under alkaline conditions (pH = 13.50), the two functional groups of thiol and acid are simultaneously adsorbed on the surface of Ag-D, while NH2 is far from the surface; under acidic conditions (pH = 1.67), adsorption behavior suggests that both NH3+ and COO- are oriented toward the Ag-D surface, and that SH is far from the surface. The structure of L-cysteine adsorption under acidic conditions can be further verified by the addition of an L-cysteine molecule through light-induced coupling reaction to form cystine. Finally, in-situ two-dimensional Raman scattering spectroscopy confirmed the feasibility and uniformity of the coupling reaction.

Measurement of Hanatoxin-Induced Membrane Thinning with Lamellar X-ray Diffraction.

Membrane perturbation induced by cysteine-rich peptides is a crucial biological phenomenon but scarcely investigated, in particular with effective biophysical-chemical methodologies. Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, works as an inhibitor of drk1 (Kv2.1) channels, most likely by interacting with the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage sensor was proposed to be deeply embedded within the bilayer. To see how HaTx interacts with phospholipid bilayers, we observe the toxin-induced perturbation on POPC/DOPG-membranes through measurements of the change in membrane thickness. Lamellar X-ray diffraction (LXD) was applied on stacked planar bilayers in the near-fully hydrated state. The results provide quantitative evidence for the membrane thinning in a concentration-dependent manner, leading to novel and direct combinatory approaches by discovering how to investigate such a biologically relevant interaction between gating-modifier toxins and phospholipid bilayers.

Supramolecular Nanostructure Formation of Coassembled Amyloid Inspired Peptides.

Characterization of amyloid-like aggregates through converging approaches can yield deeper understanding of their complex self-assembly mechanisms and the nature of their strong mechanical stability, which may in turn contribute to the design of novel supramolecular peptide nanostructures as functional materials. In this study, we investigated the coassembly kinetics of oppositely charged short amyloid-inspired peptides (AIPs) into supramolecular nanostructures by using confocal fluorescence imaging of thioflavin T binding, turbidity assay and in situ small-angle X-ray scattering (SAXS) analysis. We showed that coassembly kinetics of the AIP nanostructures were consistent with nucleation-dependent amyloid-like aggregation, and aggregation behavior of the AIPs was affected by the initial monomer concentration and sonication. Moreover, SAXS analysis was performed to gain structural information on the size, shape, electron density, and internal organization of the coassembled AIP nanostructures. The scattering data of the coassembled AIP nanostructures were best fitted into to a combination of polydisperse core-shell cylinder (PCSC) and decoupling flexible cylinder (FCPR) models, and the structural parameters were estimated based on the fitting results of the scattering data. The stability of the coassembled AIP nanostructures in both fiber organization and bulk viscoelastic properties was also revealed via temperature-dependent SAXS analysis and oscillatory rheology measurements, respectively.

The Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-Cell Transmission Properties.

Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)15 DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)15 with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-ß sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)15 DPR. Overall, our results show the structural and toxicity properties of GA DPR to facilitate future DPR-related therapeutic development.

Molecular packing and electronic processes in amorphous-like polymer bulk heterojunction solar cells with fullerene intercalation.

The interpenetrating morphology formed by the electron donor and acceptor materials is critical for the performance of polymer:fullerene bulk heterojunction (BHJ) photovoltaic (PV) cells. In this work we carried out a systematic investigation on a high PV efficiency (>6%) BHJ system consisting of a newly developed 5,6-difluorobenzo[c] thiadiazole-based copolymer, PFBT-T20TT, and a fullerene derivative. Grazing incidence X-ray scattering measurements reveal the lower-ordered nature of the BHJ system as well as an intermixing morphology with intercalation of fullerene molecules between the PFBT-T20TT lamella. Steady-state and transient photo-induced absorption spectroscopy reveal ultrafast charge transfer (CT) at the PFBT-T20TT/fullerene interface, indicating that the CT process is no longer limited by exciton diffusion. Furthermore, we extracted the hole mobility based on the space limited current (SCLC) model and found that more efficient hole transport is achieved in the PFBT-T20TT:fullerene BHJ as compared to pure PFBT-T20TT, showing a different trend as compared to the previously reported highly crystalline polymer:fullerene blend with a similar intercalation manner. Our study correlates the fullerene intercalated polymer lamella morphology with device performance and provides a coherent model to interpret the high photovoltaic performance of some of the recently developed weakly-ordered BHJ systems based on conjugated polymers with branched side-chain.

Peptide-induced bilayer thinning structure of unilamellar vesicles and the related binding behavior as revealed by X-ray scattering.

We have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bilayer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer associates closely with peptide concentration in solution and saturates at ~4%, compared to the ~8% maximum thinning observed for the correspondingly premixed peptide-MLM bilayers. Linearly scaling the thinning of peptide-ULV bilayers to that of the corresponding peptide-MLM of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant of melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy-Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC share a same thinning constant upon binding of a hydrophobic peptide alamethicin; this result supports the linear scaling approach used in the melittin-ULV bilayer thinning for thermodynamic binding parameters of water-soluble peptides.

DNA-induced aggregation of zwitterionic oligolamellar liposome.

Liposome consisting of a single zwitterionic lipid as the potential vector for gene therapy has been reported recently; however, whether polyanionic DNA can bind directly with zwitterionic lipid without the aid of multivalent salt still remains unresolved. In this study, we reveal the aggregation of zwitterionic oligolamellar liposomes composed of 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine induced by DNA without the presence of multivalent salt. Our results demonstrate that only a small fraction (<10%) of DNA can bind electrostatically with a portion of the liposomes. Such a low degree of binding, however, induces significant aggregation of these oligolamellar liposomes, yielding large multilamellar particles in which the number of hydrophilic/hydrophobic layer stacking becomes sufficiently large to yield multiple diffraction peaks in the small-angle X-ray scattering profile. Addition of monovalent salt such as NaCl tends to disrupt the multilamellar structure.