Nickel as a modifier of calcium oxalate: an in situ liquid cell TEM investigation of nucleation and growth.

Despite extensive research on the nucleation and growth of calcium oxalate (CaOx) crystals, there are still several challenges and unknowns that remain. In particular, the role of trace metal elements in the promotion or inhibition of CaOx crystals is not well understood. In the present study, in situ graphene liquid cell transmission electron microscopy (in situ GLC TEM) was used to observe real-time, nanoscale transformations of CaOx crystals in the presence of nickel ions (Ni2+). The results showed that Ni2+ form Ni-water complexes, acting as a shape-directing species, generating a unique morphology and altering growth kinetics. Transient adsorption of Ni-water complexes resulted in a metastable phase formation of calcium oxalate trihydrate. Atomistic molecular dynamics simulations confirmed that Ni2+ acts as a weak inhibitor which slows down the CaOx crystallization, elucidating that Ni2+ impacts small-sized CaOx clusters by bringing more water into the clusters. This work highlighted the intricacies behind the effect of Ni2+ on CaOx biomineralization that were made possible to discern using in situ GLC TEM.

Antiviral fibrils of self-assembled peptides with tunable compositions.

The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized ß-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

Dendrimer-Peptide Conjugates for Effective Blockade of the Interactions between SARS-CoV-2 Spike Protein and Human ACE2 Receptor.

The coronavirus disease 2019 (COVID-19) pandemic has threatened the stability of global healthcare, which is becoming an endemic issue. Despite the development of various treatment strategies to fight COVID-19, the currently available treatment options have shown varied efficacy. Herein, we have developed an avidity-based SARS-CoV-2 antagonist using dendrimer-peptide conjugates (DPCs) for effective COVID-19 treatment. Two different peptide fragments obtained from angiotensin-converting enzyme 2 (ACE2) were integrated into a single sequence, followed by the conjugation to poly(amidoamine) (PAMAM) dendrimers. We hypothesized that the strong multivalent binding avidity endowed by dendrimers would help peptides effectively block the interaction between SARS-CoV-2 and ACE2, and this antagonist effect would be dependent upon the generation (size) of the dendrimers. To assess this, binding kinetics of the DPCs prepared from generation 4 (G4) and G7 PAMAM dendrimers to spike protein of SARS-CoV-2 were quantitatively measured using surface plasmon resonance. The larger dendrimer-based DPCs exhibited significantly enhanced binding strength by 3 orders of magnitude compared to the free peptides, whereas the smaller one showed a 12.8-fold increase only. An in vitro assay using SARS-CoV-2-mimicking microbeads also showed the improved SARS-CoV-2 blockade efficiency of the G7-peptide conjugates compared to G4. In addition, the interaction between the DPCs and SARS-CoV-2 was analyzed using molecular dynamics (MD) simulation, providing an insight into how the dendrimer-mediated multivalent binding effect can enhance the SARS-CoV-2 blockade. Our findings demonstrate that the DPCs having strong binding to SARS-CoV-2 effectively block the interaction between ACE2 and SARS-CoV-2, providing a potential as a high-affinity drug delivery system to direct anti-COVID payloads to the virus.

Linear One-Dimensional Assembly of Metal Nanostructures onto an Asymmetric Peptide Nanofiber with High Persistence Length.

Self-assembled peptide fibrils have been used extensively to template the organization of metal nanoparticles in a one-dimensional (1D) array. It has been observed that the formation of the 1D arrays with a width of a single or few nanoparticles (viz. 20 nm diameter) is only possible if the templating fibers have comparable diameters (viz. ≤20 nm). Accordingly, until today, all the peptide-based templates enabling such 1D arrays have very low persistence lengths, a property that depends strongly on the diameter of the template, owing to the inherent flexibility of only a few nanometer-wide fibers. Here, we demonstrate the formation of high persistence length 1D arrays templated by a short self-assembling peptide fibril with an asymmetrically distributed charged surface. The asymmetric nature of the peptide fibril allows charge-dependent deposition of the nanoparticles only to the part of the fiber with complementary charges, and the rest of the fibril surface remains free of nanoparticles. Consequently, fibers with a much higher diameter, which will have a higher persistence length, are able to template single or few nanoparticle-wide 1D arrays. Detailed microscopy, molecular dynamics simulations, and crystal structure analysis provide molecular-level insights into fiber asymmetry and its interactions with diverse nanostructures such as gold and magnetic nanoparticles. This study will afford an alternative paradigm for high persistence length 1D array fabrication comparable to DNA nanotechnology and lithography but with tremendous cost-effectiveness.

Bimodal liquid biopsy for cancer immunotherapy based on peptide engineering and nanoscale analysis.

Despite its high potential, PD-L1 expressed by tumors has not been successfully utilized as a biomarker for estimating treatment responses to immunotherapy. Circulating tumor cells (CTCs) and tumor-derived exosomes that express PD-L1 can potentially be used as biomarkers; however, currently available assays lack clinically significant sensitivity and specificity. Here, a novel peptide-based capture surface is developed to effectively isolate PD-L1-expressing CTCs and exosomes from human blood. For the effective targeting of PD-L1, this study integrates peptide engineering strategies to enhance the binding strength and specificity of a ß-hairpin peptide derived from PD-1 (pPD-1). Specifically, this study examines the effect of poly(ethylene glycol) spacers, the secondary peptide structure, and modification of peptide sequences (e.g., removal of biologically redundant amino acid residues) on capture efficiency. The optimized pPD-1 configuration captures PD-L1-expressing tumor cells and tumor-derived exosomes with 1.5-fold (p = 0.016) and 1.2-fold (p = 0.037) higher efficiencies, respectively, than their whole antibody counterpart (aPD-L1). This enhanced efficiency is translated into more clinically significant detection of CTCs (1.9-fold increase; p = 0.035) and exosomes (1.5-fold increase; p = 0.047) from patients' baseline samples, demonstrating stronger correlation with patients' treatment responses. Additionally, we confirmed that the clinical accuracy of our system can be further improved by co-analyzing the two biomarkers (bimodal CTC/exosome analysis). These data demonstrate that pPD-1-based capture is a promising approach for capturing PD-L1-expressing CTCs and exosomes, which can be used as a reliable biomarker for cancer immunotherapy.

Creep Resistance of S304H Austenitic Steel Processed by High-Pressure Sliding.

Sheets of coarse-grained S304H austenitic steel were processed by high-pressure sliding (HPS) at room temperature and a ultrafine-grained microstructure with a mean grain size of about 0.14 µm was prepared. The microstructure changes and creep behavior of coarse-grained and HPS-processed steel were investigated at 500-700 °C under the application of different loads. It was found that the processing of S304H steel led to a significant improvement in creep strength at 500 °C. However, a further increase in creep temperature to 600 °C and 700 °C led to the deterioration of creep behavior of HPS-processed steel. The microstructure results suggest that the creep behavior of HPS-processed steel is associated with the thermal stability of the SPD-processed microstructure. The recrystallization, grain growth, the coarsening of precipitates led to a reduction in creep strength of the HPS-processed state. It was also observed that in the HPS-processed microstructure the fast formation of σ-phase occurs. The σ-phase was already formed during slight grain coarsening at 600 °C and its formation was enhanced after recrystallization at 700 °C.

Hierarchically Multivalent Peptide-Nanoparticle Architectures: A Systematic Approach to Engineer Surface Adhesion.

The multivalent binding effect has been the subject of extensive studies to modulate adhesion behaviors of various biological and engineered systems. However, precise control over the strong avidity-based binding remains a significant challenge. Here, a set of engineering strategies are developed and tested to systematically enhance the multivalent binding of peptides in a stepwise manner. Poly(amidoamine) (PAMAM) dendrimers are employed to increase local peptide densities on a substrate, resulting in hierarchically multivalent architectures (HMAs) that display multivalent dendrimer-peptide conjugates (DPCs) with various configurations. To control binding behaviors, effects of the three major components of the HMAs are investigated: i) poly(ethylene glycol) (PEG) linkers as spacers between conjugated peptides; ii) multiple peptides on the DPCs; and iii) various surface arrangements of HMAs (i.e., a mixture of DPCs each containing different peptides vs DPCs cofunctionalized with multiple peptides). The optimized HMA configuration enables significantly enhanced target cell binding with high selectivity compared to the control surfaces directly conjugated with peptides. The engineering approaches presented herein can be applied individually or in combination, providing guidelines for the effective utilization of biomolecular multivalent interactions using DPC-based HMAs.

An Organometallic Strategy for Cysteine Borylation.

Synthetic bioconjugation at cysteine (Cys) residues in peptides and proteins has emerged as a powerful tool in chemistry. Soft nucleophilicity of the sulfur in Cys renders an exquisite chemoselectivity with which various functional groups can be placed onto this residue under benign conditions. While a variety of reactions have been successful at producing Cys-based bioconjugates, the majority of these feature sulfur-carbon bonds. We report Cys-borylation, wherein a benchtop stable Pt(II)-based organometallic reagent can be used to transfer a boron-rich cluster onto a sulfur moiety in unprotected peptides forging a boron-sulfur bond. Cys-borylation proceeds at room temperature and tolerates a variety of functional groups present in complex polypeptides. Further, the bioconjugation strategy can be applied to a model protein modification of Cys-containing DARPin (designed ankyrin repeat protein). The resultant bioconjugates show no additional toxicity compared to their Cys alkyl-based congeners. Finally, we demonstrate how the developed Cys-borylation can enhance the proteolytic stability of the resultant peptide bioconjugates while maintaining the binding affinity to a protein target.

Hybridization of Biomolecular Crystals and Low-Dimensional Materials.

In cellular environments, metabolites, peptides, proteins, and other biomolecules can self-assemble into planar and fibrilar molecular crystals. We use atomistic molecular dynamics simulations to show that such biomolecular crystals coupled with low-dimensional materials can form stable hybrid superstructures. We discuss enantiopure and racemic TRP and PHE amino acid crystals adsorbed on or intercalated between graphene, phosphorene, and carbon nanotubes. While racemic biomolecular crystals tend to stay straight in solutions and when adsorbed on flat and cylindrical nanosurfaces, enantiopure crystals undergo twisting. Mixed material properties of these hybrid superstructures can be attractive in many applications.

Retrained Generic Antibodies Can Recognize SARS-CoV-2.

The dramatic impact novel viruses can have on humans could be more quickly mitigated if generic antibodies already present in one's system are temporarily retrained to recognize these viruses. This type of intervention can be administered during the early stages of infection, while a specific immune response is being developed. With this idea in mind, double-faced peptide-based boosters were computationally designed to allow recognition of SARS-CoV-2 by Hepatitis B antibodies. One booster face is made of ACE2-mimic peptides that can bind to the receptor binding domain (RBD) of SARS-CoV-2. The other booster face is composed of a Hepatitis B core-antigen, targeting the Hepatitis B antibody fragment. Molecular dynamics simulations revealed that the designed boosters have a highly specific and stable binding to both the RBD and the antibody fragment (AF). This approach can provide a cheap and efficient neutralization of emerging pathogens.

The Effect of Predeformation on Creep Strength of 9% Cr Steel.

Martensitic creep-resistant P92 steel was deformed by different methods of severe plastic deformation such as rotation swaging, high-pressure sliding, and high-pressure torsion at room temperature. These methods imposed significantly different equivalent plastic strains of about 1-30. It was found that rotation swaging led to formation of heterogeneous microstructures with elongated grains where low-angle grain boundaries predominated. Other methods led to formation of ultrafine-grained (UFG) microstructures with high frequency of high-angle grain boundaries. Constant load tensile creep tests at 873 K and initial stresses in the range of 50 to 300 MPa revealed that the specimens processed by rotation swaging exhibited one order of magnitude lower minimum creep rate compared to standard P92 steel. By contrast, UFG P92 steel is significantly softer than standard P92 steel, but differences in their strengths decrease with increasing stress. Microstructural results suggest that creep behavior of P92 steel processed by severe plastic deformation is influenced by the frequency of high-angle grain boundaries and dynamic grain coarsening during creep.

Tuning of the Aggregation Behavior of Fluorinated Polymeric Nanoparticles for Improved Therapeutic Efficacy.

Incorporation of fluorinated moieties in polymeric nanoparticles has been shown in many instances to increase their uptake by living cells and, hence, has proven to be a useful approach to enhancing delivery to cells. However, it remains unclear how incorporation of fluorine affects critical transport processes, such as interactions with membranes, intracellular transport, and tumor penetration. In this study, we investigate the influence of fluorine on transport properties using a series of rationally designed poly(oligo(ethylene glycol) methyl ether acrylate)-block-perfluoropolyether (poly(OEGA)m-PFPE) copolymers. Copolymers with different fluorine contents were prepared and exhibit aggregate in solution in a manner dependent on the fluorine content. Doxorubicin-conjugated poly(OEGA)20-PFPE nanoparticles with lower fluorine content exist in solution as unimers, leading to greater exposure of hydrophobic PFPE segments to the cell surface. This, in turn, results in greater cellular uptake, deeper tumor penetration, as well as enhanced therapeutic efficacy compared to that with the micelle-state nanoaggregates (poly(OEGA)10-PFPE and poly(OEGA)5-PFPE) with higher fluorine content but with less PFPE exposed to the cell membranes. Our results demonstrate that the aggregation behavior of these fluorinated polymers plays a critical role in internalization and transport in living cells and 3D spheroids, providing important design criteria for the preparation of highly effective delivery agents.

Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2.

Peptide inhibitors against the SARS-CoV-2 coronavirus, currently causing a worldwide pandemic, are designed and simulated. The inhibitors are mostly formed by two sequential self-supporting α-helices (bundle) extracted from the protease domain (PD) of angiotensin-converting enzyme 2 (ACE2), which bind to the SARS-CoV-2 receptor binding domains. Molecular dynamics simulations revealed that the α-helical peptides maintain their secondary structure and provide a highly specific and stable binding (blocking) to SARS-CoV-2. To provide a multivalent binding to the SARS-CoV-2 receptors, many such peptides could be attached to the surfaces of nanoparticle carriers. The proposed peptide inhibitors could provide simple and efficient therapeutics against the COVID-19 disease.

Nanoparticle Conjugation Stabilizes and Multimerizes ß-Hairpin Peptides To Effectively Target PD-1/PD-L1 ß-Sheet-Rich Interfaces.

ß-Hairpin peptides present great potential as antagonists against ß-sheet-rich protein surfaces, of which wide and flat geometries are typically "undruggable" with small molecules. Herein, we introduce a peptide-dendrimer conjugate (PDC) approach that stabilizes the ß-hairpin structure of the peptide via intermolecular forces and the excluded volume effect as well as exploits the multivalent binding effect. Because of the synergistic advantages, the PDCs based on a ß-hairpin peptide isolated from an engineered programmed death-1 (PD-1) protein showed significantly higher affinity (avidity) to their binding counterpart, programmed death-ligand 1 (PD-L1), as compared to free peptides (by up to 5 orders of magnitude). The enhanced binding kinetics with high selectivity was translated into an improved immune checkpoint inhibitory effect in vitro, at a level comparable to (if not better than) that of a full-size monoclonal antibody. The results demonstrate the potential of the PDC system as a novel class of inhibitors targeting ß-strand-rich protein surfaces, such as PD-1 and PD-L1, displaying its potential as a new cancer immunotherapy platform.

Assessment of Retained Austenite in Fine Grained Inductive Heat Treated Spring Steel.

Advanced thermomechanical hot rolling is becoming a widely used technology for the production of fine-grained spring steel. Different rapid phase transformations during the inductive heat treatment of such steel causes the inhomogeneous mixture of martensitic, bainitic, and austenitic phases that affects the service properties of the steel. An important task is to assess the amount of retained austenite and its distribution over the cross-section of the inductive quenched and tempered wire in order to evaluate the mechanical properties of the material. Three different analytical methods were used for the comparative quantitative assessment of the amount of retained austenite in both the core and rim areas of the sample cross-section: neutron diffraction-for the bulk of the material, Mössbauer spectroscopy-for measurement in a surface layer, and the metallographic investigations carried by the EBSD. The methods confirmed the excessive amount of retained austenite in the core area that could negatively affect the plasticity of the material.

Novel Oligo-Guanidyl-PEG Carrier Forming Rod-Shaped Polyplexes.

A novel unconventional supramolecular oligo-cationic structure (Agm6-M-PEG-OCH3) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm6-M-PEG-OCH3 was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm6-M-PEG-OCH3 at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm6-M-PEG-OCH3/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm6-M-PEG-OCH3/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm6-M-PEG-OCH3 nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.

Electrical Conductivity, Selective Adhesion, and Biocompatibility in Bacteria-Inspired Peptide-Metal Self-Supporting Nanocomposites.

Bacterial type IV pili (T4P) are polymeric protein nanofibers that have diverse biological roles. Their unique physicochemical properties mark them as a candidate biomaterial for various applications, yet difficulties in producing native T4P hinder their utilization. Recent effort to mimic the T4P of the metal-reducing Geobacter sulfurreducens bacterium led to the design of synthetic peptide building blocks, which self-assemble into T4P-like nanofibers. Here, it is reported that the T4P-like peptide nanofibers efficiently bind metal oxide particles and reduce Au ions analogously to their native counterparts, and thus give rise to versatile and multifunctional peptide-metal nanocomposites. Focusing on the interaction with Au ions, a combination of experimental and computational methods provides mechanistic insight into the formation of an exceptionally dense Au nanoparticle (AuNP) decoration of the nanofibers. Characterization of the thus-formed peptide-AuNPs nanocomposite reveals enhanced thermal stability, electrical conductivity from the single-fiber level up, and substrate-selective adhesion. Exploring its potential applications, it is demonstrated that the peptide-AuNPs nanocomposite can act as a reusable catalytic coating or form self-supporting immersible films of desired shapes. The films scaffold the assembly of cardiac cells into synchronized patches, and present static charge detection capabilities at the macroscale. The study presents a novel T4P-inspired biometallic material.

Transition of Metastable Cross-α Crystals into Cross-ß Fibrils by ß-Turn Flipping.

The ensemble of native, folded state was once considered to represent the global energy minimum of a given protein sequence. More recently, the discovery of the cross-ß amyloid state revealed that deeper energy minima exist, often associated with pathogenic, fibrillar deposits, when the concentration of proteins reaches a critical value. Fortunately, a sizable energy barrier impedes the conversion from native to pathogenic states. However, little is known about the structure of the related transition state. In addition, there are indications of polymorphism in the amyloidogenic process. Here, we report the first evidence of the conversion of metastable cross-α-helical crystals to thermodynamically stable cross-ß-sheet-like fibrils by a de novo designed heptapeptide. Furthermore, for the first time, we demonstrate at atomic resolution that the flip of a peptide plane from a type I to a type II' turn facilitates transformation to cross-ß structure and assembly of a dry steric zipper. This study establishes the potential of a peptide turn, a common protein secondary structure, to serve as a principal gatekeeper between a native metastable folded state and the amyloid state.

High F-Content Perfluoropolyether-Based Nanoparticles for Targeted Detection of Breast Cancer by 19F Magnetic Resonance and Optical Imaging.

Two important challenges in the field of 19F magnetic resonance imaging (MRI) are the maintenance of high fluorine content without compromising imaging performance, and effective targeting of small particles to diseased tissue. To address these challenges, we have developed a series of perfluoropolyether (PFPE)-based hyperbranched (HBPFPE) nanoparticles with attached peptide aptamer as targeting ligands for specific in vivo detection of breast cancer with high 19F MRI sensitivity. A detailed comparison of the HBPFPE nanoparticles (NPs) with the previously reported trifluoroethyl acrylate (TFEA)-based polymers demonstrates that the mobility of fluorinated segments of the HBPFPE nanoparticles is significantly enhanced (19F T2 > 80 ms vs 31 ms), resulting in superior MR imaging sensitivity. Selective targeting was confirmed by auto- and pair correlation analysis of fluorescence microscopy data, in vitro immunofluorescence, in vivo 19F MRI, ex vivo fluorescence and 19F NMR. The results highlight the high efficiency of aptamers for targeting and the excellent sensitivity of the PFPE moieties for 19F MRI. Of relevance to in vivo applications, the PFPE-based polymers exhibit much faster clearance from the body than the previously introduced perfluorocarbon emulsions ( t1/2 ∼ 20 h vs up to months). Moreover, the aptamer-conjugated NPs show significantly higher tumor-penetration, demonstrating the potential of these imaging agents for therapeutic applications. This report of the synthesis of polymeric aptamer-conjugated PFPE-based 19F MRI CAs with high fluorine content (∼10 wt %) demonstrates that these NPs are exciting candidates for detecting diseases with high imaging sensitivity.