Publications

Permanently polarized biphasic calcium phosphate composed of hydroxyapatite and brushite (pp-HAp/Bru), which is prepared by applying the thermally stimulated polarization treatment to calcined HAp, is used as a sustainable catalyst to transform CO2 into value-added products. In this work, the stability of pp-HAp/Bru is studied from structural, electrical, and catalytic perspectives, applying a thermal depolarization process with temperatures (T-d) ranging from 200 to 1000 degrees C. Results show that the Bru phase is not stable when T-d = 600 degrees C. Besides, the electrical resistance and capacitance of the pp-HAp/Bru increase with T-d, evidencing the progressive electrical depolarization of the material. Thermal depolarization also influences the specific orientation of the OH- ions, which is partially lost (approximate to 50%). All such changes affect the catalytic efficiency of pp-HAp/Bru, which is proven using a reaction that transforms CO2 gas into acetic acid and formic acid. Results show that the total reaction yield linearly decreases with increasing T-d. Based on such observations, a simple process is designed that allows the reconstitution of the structure and restores the activity of such green catalysts.

JTD Keywords: Co-2 fixation, Electrical depolarization, Green catalysts, Interface stability, Thermal depolarizatio

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.© 2024 The Authors. Small Methods published by Wiley-VCH GmbH.

JTD Keywords: 3d cancer cell uptake, Cancer cells, Cell culture, Cell uptake, Cellular uptake, Diseases, Ecm penetration, Extracellular matrices, Extracellular matrix penetration, Functional polymers, Hydrogen bonds, Medical applications, Microfluidics, Microstructure, Nanoparticles, Polymeric nanoparticles, Scpns, Single chains, Single-chain polymeric nanoparticle, Stability, Tumor-on-a-chip, Tumors

Dental implant-associated infection is a clinical challenge which poses a significant healthcare and socio-economic burden. To overcome this issue, developing antimicrobial surfaces, including antimicrobial peptide coatings, has gained great attention. Different physical and chemical routes have been used to obtain these biofunctional coatings, which in turn might have a direct influence on their bioactivity and functionality. In this study, we present a silane-based, fast, and efficient chemoselective conjugation of antimicrobial peptides (Cys-GL13K) to coat titanium implant surfaces. Comprehensive surface analysis was performed to confirm the surface functionalization of as-prepared and mechanically challenged coatings. The antibacterial potency of the evaluated surfaces was confirmed against both Streptococcus gordonii and Streptococcus mutans, the primary colonizers and pathogens of dental surfaces, as demonstrated by reduced bacteria viability. Additionally, human dental pulp stem cells demonstrated long-term viability when cultured on Cys-GL13K-grafted titanium surfaces. Cell functionality and antimicrobial capability against multi-species need to be studied further; however, our results confirmed that the proposed chemistry for chemoselective peptide anchoring is a valid alternative to traditional site-unspecific anchoring methods and offers opportunities to modify varying biomaterial surfaces to form potent bioactive coatings with multiple functionalities to prevent infection.

JTD Keywords: biocompatibility, cytotoxicity, delivery, dental implants, prevention, release, stability, surface coating, titanium, zirconia, Antimicrobial peptide, Biocompatibility, Dental implants, Peri-implantitis, Surface coating, Titanium

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aβ). Numerous alternatives have been studied for inhibiting Aβ aggregation but, at this time, there are no effective treatments available. Here, we developed the tri-component nanohybrid system AuNPs@POM@PEG based on gold nanoparticles (AuNPs) covered with polyoxometalates (POMs) and polyethylene glycol (PEG). In this work, AuNPs@POM@PEG demonstrated the inhibition of the formation of amyloid fibrils, showing a 75% decrease in Aβ aggregation in vitro. As it is a potential candidate for the treatment of Alzheimer's disease, we evaluated the cytotoxicity of AuNPs@POM@PEG and its ability to cross the blood-brain barrier (BBB). We achieved a stable nanosystem that is non-cytotoxic below 2.5 nM to human neurovascular cells. The brain permeability of AuNPs@POM@PEG was analyzed in an in vitro microphysiological model of the BBB (BBB-on-a-chip), containing 3D human neurovascular cell co-cultures and microfluidics. The results show that AuNPs@POM@PEG was able to cross the brain endothelial barrier in the chip and demonstrated that POM does not affect the barrier integrity, giving the green light to further studies into this system as a nanotherapeutic.

JTD Keywords: beta-amyloid, blood-brain barrier organ-on-a-chip, cellular uptake, citrate, cytotoxicity, electrocatalytic reduction, gold nanoparticles, hypothesis, nanorods, polyoxometalates, size, stability, surface, Alzheimers-disease, Blood–brain barrier organ-on-a-chip, Gold nanoparticles, Nanovehicle, Polyoxometalates, Β-amyloid

In order to exploit the pharmacological potential of natural bioactive molecules with low water solubility, such as curcumin, it is necessary to develop formulations, such as amorphous polymer dispersions, which allow a constant release rate and at the same time avoid possible toxicity effects of the crystalline form of the molecule under scrutiny. In this study, polymer dispersions of curcumin were obtained in PADAS, a biodegradable semicrystalline copolymer based on 1,12-dodecanediol, sebacic acid and alanine. The dispersions were fully characterized by means of differential scanning calorimetry and broadband dielectric spectroscopy, and the drug release profile was measured in a simulated body fluid. Amorphous homogeneous binary dispersions were obtained for curcumin mass fraction between 30 and 50%. Curcumin has significantly higher glass transition temperature Tg (≈ 347 K) than the polymer matrix (≈274-277 K depending on the molecular weight), and dispersions displayed Tg's intermediate between those of the pure amorphous components, implying that curcumin acts as an effective antiplasticizer for PADAS. Dielectric spectroscopy was employed to assess the relaxation dynamics of the binary dispersion with 30 wt% curcumin, as well as that of each (amorphous) component separately. The binary dispersion was characterized by a single structural relaxation, a single Johari-Goldstein process, and two local intramolecular processes, one for each component. Interestingly, the latter processes scaled with the Tg of the sample, indicating that they are viscosity-sensitive. In addition, both the pristine polymer and the dispersion exhibited an interfacial Maxwell-Wagner relaxation, likely due to spatial heterogeneities associated with phase disproportionation in this polymer. The release of curcumin from the dispersion in a simulated body fluid followed a Fickian diffusion profile, and 51% of the initial curcumin content was released in 48 h.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: antioxidant, bioavailability, dielectric spectroscopy, domain havriliak-negami, glass transition temperature, kinetic stability, molecular mobility, nm pores, phase-behavior, physical stability, release kinetics, temperature, thermodynamic quantities, time, Amorphous formulations, Dielectric spectroscopy, Glass transition temperature, Kinetic stability, Kohlrausch-williams-watts, Molecular mobility, Release kinetics

Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (≤168 nm) and polydispersity indexes (≤0.16) and ζ-potentials (≤-35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56-78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs.

JTD Keywords: biodegradation, copolymer ratio, degradation, drug-delivery, emulsification, enzyme release, enzyme replacement therapy, hyaluronidase, mechanisms, microspheres, nanoparticle stability, poly(lactide-co-glycolide) nanoparticles, size, sphingomyelinase, transport, Central-nervous-system, Copolymer ratio, Enzyme release, Enzyme replacement therapy, Hyaluronidase, Lysosomal storage disorder, Nanoparticle stability, Poly(lactide-co-glycolide) nanoparticles

JTD Keywords: alpha-tricalcium phosphate, cement, crystallinity, hydrolysis, hydroxyapatite, particle-size, powders, relative humidity, setting reaction, stability, tcp, vapor sorption, Calcium-phosphate, Moisture

Polymeric spherical nucleic acids comprise pH-sensitive, polymer-conjugated oligonucleotides that self-assemble into nanoparticles with the ability to escape endosomes, overcoming a major obstacle in nucleic acid delivery.

JTD Keywords: c9orf72, cellular uptake, dna, encapsulation, expansion, ph, stability, trafficking, vesicles, Hexanucleotide repeat

Mechanical force modulates the conformation and function of individual proteins, and this underpins many mechanically driven cellular processes. This Review addresses single-molecule force spectroscopy experiments conducted on proteins with a known role in mechanosensing and mechanotransduction in eukaryotic cells.; In addition to biochemical signals and genetic considerations, mechanical forces are rapidly emerging as a master regulator of human physiology. However, the molecular mechanisms that regulate force-induced functionalities across a wide range of scales, encompassing the cell, tissue or organ levels, are not well understood in comparison. With the advent, development and refining of single-molecule nanomechanical techniques that enable the conformational dynamics of individual proteins under the effect of a calibrated force to be probed, we have begun to acquire a comprehensive knowledge of the diverse physicochemical principles that regulate the elasticity of single proteins. Here, we review the major advances underpinning our current understanding of how the elasticity of single proteins regulates mechanosensing and mechanotransduction. We discuss the present limitations and future challenges of this prolific and burgeoning field.

JTD Keywords: Cadherin adhesion, Energy landscape, Extracellular-matrix protein, Focal adhesion kinase, Mechanical stability, Molecule force spectroscopy, Muscle protein, N2b element, Stranded-dna, Structural basis

Building functional mimics of cell membranes is an important task toward the development of synthetic cells. So far, lipid and amphiphilic block copolymers are the most widely used amphiphiles with the bilayers by the former lacking stability while membranes by the latter are typically characterized by very slow dynamics. Herein, we introduce a new type of Janus dendrimer containing a zwitterionic phosphocholine hydrophilic headgroup (JDPC ) and a 3,5-substituted dihydrobenzoate-based hydrophobic dendron. JDPC self-assembles in water into zwitterionic dendrimersomes (z-DSs) that faithfully recapitulate the cell membrane in thickness, flexibility, and fluidity, while being resilient to harsh conditions and displaying faster pore closing dynamics in the event of membrane rupture. This enables the fabrication of hybrid DSs with components of natural membranes, including pore-forming peptides, structure-directing lipids, and glycans to create raft-like domains or onion vesicles. Moreover, z-DSs can be used to create active synthetic cells with life-like features that mimic vesicle fusion and motility as well as environmental sensing. Despite their fully synthetic nature, z-DSs are minimal cell mimics that can integrate and interact with living matter with the programmability to imitate life-like features and beyond. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

JTD Keywords: biological-membranes, bottom-up synthetic biology, chain, hybrid vesicles, hydroethidine, organization, polymersome, proteins, stability, synthetic cells, thickness, vesicle fusion, vesicle motility, vesicles, zwitterionic dendrimersomes, Biosensor, Biosensors, Bottom-up synthetic biology, Hybrid vesicles, Lipid-bilayers, Synthetic cells, Vesicle fusion, Vesicle motility, Zwitterionic dendrimersomes

Hydrogels have been recently proposed as suitable materials to generate reactive oxygen and nitrogen species (RONS) upon gas-plasma treatment, and postulated as promising alternatives to conventional cancer therapies. Acting as delivery vehicles that allow a controlled release of RONS to the diseased site, plasma-treated hydrogels can overcome some of the limitations presented by plasma-treated liquids in in vivo therapies. In this work, we optimized the composition of a methylcellulose (MC) hydrogel to confer it with the ability to form a gel at physiological temperatures while remaining in the liquid phase at room temperature to allow gas-plasma treatment with suitable formation of plasma-generated RONS. MC hydrogels demonstrated the capacity for generation, prolonged storage and release of RONS. This release induced cytotoxic effects on the osteosarcoma cancer cell line MG-63, reducing its cell viability in a dose-response manner. These promising results postulate plasma-treated thermosensitive hydrogels as good candidates to provide local anticancer therapies.

JTD Keywords: Case-control studies, Cellulose, Hydrogels, Methylcellulose, Phase-separation, Plasma gases, Reactive oxygen species, Stability, Substituent, Temperature, Thermoreversible gelation

Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(?-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.

JTD Keywords: cancer nanomedicine, cell trafficking, delivery, direct stochastic optical reconstruction microscopy (dstorm), nanoparticle stability, poly(beta-aminoester) nanoparticles, Direct stochastic optical reconstruction microscopy (dstorm), Poly(?-aminoester) nanoparticles, Poly(beta-amino ester)s, Poly(β-aminoester) nanoparticles

Tuning of the crosslink density (CLD) in the rubber compounds is very crucial for optimizing the physical and mechanical properties of the ultimate rubber products. Conventionally, CLD can be measured via rheological methods such as moving die rheometer (MDR), via mechanical tests such as temperature scanning stress relaxation analysis (TSSR), or via direct swelling experiments using Flory–Rehner approach. In the current study, two novel techniques, focused ion beam - scanning electron microscopy (FIB-SEM) processing, with simultaneous energy dispersive X-ray spectrometry (EDS) mapping analysis and scanning acoustic microscopy (SAM) were combined and correlated to conventional methods on a model recipe of ethylene propylene diene monomer (EPDM) compound having different sulphur contents. Depending on the applied technique, the increase in the crosslink density with sulphur content was found to be 1.7 fold for the Flory–Rehner approach and 1.2 fold for both TSSR and MDR. It is directly monitored from the FIB-SEM-EDS analysis that the sulphur distribution and agglomeration behavior increased in line with ZnO content, which is an indirect indication of the rise in crosslink density. The impedance maps of the crosslinked samples obtained through SAM analysis revealed that the impedance of the samples increased with the increasing sulphur content, which can be attributed to higher level of crosslink density. A quantified correlation was obtained between SAM images and the crosslink density of the samples. It was shown that SAM is a promising tool for practical and non-destructive analysis for determining the formation of crosslink density of the rubbers. © The Author(s) 2022.

JTD Keywords: blends, compressibility, crosslink density, cure characteristics, ethylene propylene diene monomer, focused ion beam, mechanical-properties, morphology, natural-rubber, particles, scanning acoustic microscopy, scanning electron microscopy, sulfur, thermal-stability, vulcanization, Composite soft materials, Cross-link densities, Crosslink density, Crosslinking, Density (specific gravity), Ethylene, Ethylene propylene diene monomer, Flory-rehner, Focused ion beam - scanning electron microscopy, Focused ion beam-scanning electron microscopies, Ii-vi semiconductors, Monomers, Moving die rheometers, Physical and mechanical properties, Propylene, Relaxation analysis, Rubber, Scanning acoustic microscopy, Scanning electron microscopy, Stress relaxation, Sulfur contents, Temperature scanning stress relaxations, Zinc oxide

Objective. Impaired trunk stability is frequent in spinal cord injury (SCI), but there is a lack of quantitative measures for assessing trunk function. Our objectives were to: (a) evaluate trunk muscle activity and movement patterns during a reaching task in SCI patients, (b) compare the impact of cervical (cSCI) and thoracic (tSCI) injuries in trunk function, and (c) investigate the effects of a startling acoustic stimulus (SAS) in these patients. Approach. Electromyographic (EMG) and smartphone accelerometer data were recorded from 15 cSCI patients, nine tSCI patients, and 24 healthy controls, during a reaching task requiring trunk tilting. We calculated the response time (RespT) until pressing a target button, EMG onset latencies and amplitudes, and trunk tilt, lateral deviation, and other movement features from accelerometry. Statistical analysis was applied to analyze the effects of group (cSCI, tSCI, control) and condition (SAS, non-SAS) in each outcome measure. Main results. SCI patients, especially those with cSCI, presented significantly longer RespT and EMG onset latencies than controls. Moreover, in SCI patients, forward trunk tilt was accompanied by significant lateral deviation. RespT and EMG latencies were remarkably shortened by the SAS (the so-called StartReact effect) in tSCI patients and controls, but not in cSCI patients, who also showed higher variability. Significance. The combination of EMG and smartphone accelerometer data can provide quantitative measures for the assessment of trunk function in SCI. Our results show deficits in postural control and compensatory strategies employed by SCI patients, including delayed responses and higher lateral deviations, possibly to improve sitting balance. This is the first study investigating the StartReact responses in trunk muscles in SCI patients and shows that the SAS significantly accelerates RespT in tSCI, but not in cSCI, suggesting an increased cortical control exerted by these patients.

JTD Keywords: accelerometer, electromyography, impairment, individuals, movements, postural stability, reaction-time, reliability, sitting balance, smartphone, spinal cord injury, startle, startreact, strategies, stroke, trunk, Accelerometer, Electromyography, Sitting balance, Smartphone, Spinal cord injury, Startreact, Trunk

A new methodology for the pH-triggered degradation of polymers or for the release of drugs under visible light irradiation based on the cyclization of ortho-hydroxy-cinnamates (oHC) to coumarins is described. The key oHC structural motif can be readily incorporated into the rational design of novel photocleavable polymers via click chemistry. This main-chain moiety undergoes a fast photocleavage when irradiated with 455 nm light provided that a suitable base is added. A series of polyethylene glycol-alt-ortho-hydroxy cinnamate (polyethylene glycol (PEG)(n)-alt-oHC)-based polymers are synthesized and the time-dependent visible-light initiated cleavage of the photoactive monomer and polymer is investigated in solution by a variety of spectroscopic and chromatographic techniques. The photo-degradation behavior of the water-soluble poly(PEG(2000)-alt-oHC) is investigated within a broad pH range (pH = 2.1-11.8), demonstrating fast degradation at pH 11.8, while the stability of the polymer is greatly enhanced at pH 2.1. Moreover, the neat polymer shows long-term stability under daylight conditions, thus allowing its storage without special precautions. In addition, two water-soluble PEG-based drug-carrier molecules (mPEG(2000)-oHC-benzhydrol/phenol) are synthesized and used for drug delivery studies, monitoring the process by UV-vis spectroscopy in an ON/OFF intermittent manner.

JTD Keywords: coumarins, drug delivery, e/z-double bond isomerization, o-hydroxy cinnamates, polymer degradation, Aliphatic compounds, Antioxidant activity, Antitumor, Chromatographic techniques, Chromatography, Cis/trans isomerization, Controlled drug delivery, Coumarin derivatives, Coumarins, Drug delivery, Drug delivery system, E/z-double bond isomerization, Films, Hydrogels, Image enhancement, Light, Long term stability, O-hydroxy cinnamates, Particles, Photoactive monomers, Photodegradation, Polyethylene glycols, Polyethylenes, Polymer degradation, Responsive polymers, Salts, Structural motifs, Synthesis (chemical), Targeted drug delivery, Visible light photocatalysis, Visible-light irradiation

© 2020 American Chemical Society. The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

JTD Keywords: cancer-on-a-chip, complex, delivery, endothelial-cells, in-vitro, microfluidic, model, nanoparticle, penetration, shear-stress, stability, supramolecular, Cancer-on-a-chip, Cell-culture, Micelle, Microfluidic, Nanoparticle, Stability, Supramolecular

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

JTD Keywords: dSTORM, Gene delivery, Polyplexes, Stability, Super-resolution microscopy

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation. Force-dependent changes in nuclear pores control protein access to the nucleus.

JTD Keywords: Atomic force microscopy, Hippo pathway, Mechanosensing, Mechanotransduction, Molecular mechanical stability, Nuclear mechanics, Nuclear pores, Nuclear transport, Rigidity sensing, Transcription regulation

Graft polymer surfactants provide very good colloidal stability because of strong steric repulsions between adsorbed surfactant films. The elastic and adhesion properties of adsorbed hydrophobically modified inulin polymer surfactant (INUTEC NRA) have been directly measured using Atomic Force Microscopy (AFM) measurements. For this purpose, poly(methyl methacrylate/butyl acrylate), P(MMA/BuA), latexes prepared in the presence of the hydrophobically modified inulin (INUTEC NRA) were used. These latexes (diameter 118 nm and polydispersity index of 1.05) showed a very high colloidal stability in water and in the presence of electrolyte (up to 0.2 mol dm−3 KBr). The latexes were deposited on mica, which was silanated to enhance the adhesion of the latex particles to the surface. A silicon nitride tip with approximately 10 nm diameter that also contained an adsorbed layer of surfactant was used in the AFM apparatus. The tip was allowed to approach, contact thereafter the particles with an applied force of 12.5 nN, and finally detach from the film. Both elastic (Young’s) modulus of the film and adhesion force were studied. The results showed that the adsorbed surfactant films are highly elastic and their elastic modulus and adhesion force did not change significantly with the presence of Na2SO4 up to 0.05 mol dm−3. The high elastic contribution to the steric interaction ensures strong repulsion between the latex particles both in water and at high electrolyte concentrations. In addition, the lack of dependence of adhesion force on electrolyte concentration ensures uniform deposition of the latex particles on a flat substrate as for example in coating applications. These results show the advantages of using a graft polymer surfactant for enhancing the stability of particle suspensions, as illustrated in previous investigations.

JTD Keywords: AFM, Colloidal stability, Interaction forces, Steric repulsion

IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC-E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance.

JTD Keywords: Antimicrobial resistance, E protein, IncC, IncHI1 plasmids, Plasmid R27, Plasmid stability

Abstract Nanocomposite powders composed by polymeric micelles as vehicles for delivery proteins were developed in this work, using insulin as model protein. Results showed that size and polydispersity of micelles were dependent on the amphiphilic polymer used, being all lower than 300 nm, while all the formulations displayed spherical shape and surface charge close to neutrality. Percentages of association efficiency and loading capacity up to 94.15 ± 3.92 and 8.56 ± 0.36, respectively, were obtained. X-ray photoelectron spectroscopy (XPS) measurements confirmed that insulin was partially present at the hydrophilic shell of the micelles. Lyophilization did not significantly change the physical characteristics of micelles, further providing easily dispersion when in contact to aqueous medium. The native-like conformation of insulin was maintained at high percentages (around 80%) after lyophilization as indicated by Fourier transform infrared spectroscopy (FTIR) and far-UV circular dichroism (CD). Moreover, Raman spectroscopy did not evidenced significant interactions among the formulation components. The formulations shown to be physically stable upon storage up to 6 months both at room-temperature (20 C) and fridge (4 C), with only a slight loss (maximum of 15%) of the secondary structure of the protein. Among the polymers tested, Pluronic® F127 produced the carrier formulations more promising for delivery of proteins.

JTD Keywords: Amphiphilic polymers, Insulin, Lyophilization, Polymeric micelles, Stability

Abstract Wearable robots are expected to expand the use of robotics in rehabilitation since they can widen the assistance application context. An important aspect of a rehabilitation therapy, in terms of lower extremity assistance, is balance control. In this article, we propose and evaluate an adaptive control strategy for robotic rehabilitation therapies to guarantee static stability using a wearable robot. Postural balance control can be implemented either acting on the hip, on the ankle joint or on both, depending on the kind of perturbation acting on the subject: internal or external. Internal perturbations can be produced by any voluntary movement of the body, such as bending the trunk. External perturbations, in the form of an impact force, are applied by the exoskeleton without any prior notice to observe the proactive response of the subject. We have used a 6 degree of freedom planar lower limb exoskeleton, H1, to perform this analysis. The developed control strategy has been designed to provide the necessary assistance, related to balance recovery and postural stability, under the “Assist-as-needed” paradigm. The interaction forces between orthosis and subject are monitored, as they play a relevant role in the definition of assistive and resistive movements to be applied to the joints. The proposed method has been tested with 5 healthy subjects in presence of internal and external disturbances. The results demonstrate that knowing the stability limit of each subject, in combination with a therapeutically selected scaling factor, the proposed adaptive control helps in providing an effective assistance in therapy. This method is efficient in handling the individual and combined effect of external perturbations acting on any joint movements.

JTD Keywords: Exoskeleton controls, Postural stability, Balance controls, Adaptive control

Poly(vinylchloride) (PVC) is the most common polymer matrix used in the fabrication of ion-selective electrodes (ISEs). However, the surfaces of PVC-based sensors have been reported to show membrane instability. In an attempt to overcome this limitation, here we developed two alternative methods for the preparation of highly stable and robust ion-selective sensors. These platforms are based on the selective electropolymerization of poly(3,4-ethylenedioxythiophene) (PEDOT), where the sulfur atoms contained in the polymer covalently interact with the gold electrode, also permitting controlled selective attachment on a miniaturized electrode in an array format. This platform sensor was improved with the crosslinking of the membrane compounds with poly(ethyleneglycol) diglycidyl ether (PEG), thus also increasing the biocompatibility of the sensor. The resulting ISE membranes showed faster signal stabilization of the sensor response compared with that of the PVC matrix and also better reproducibility and stability, thus making these platforms highly suitable candidates for the manufacture of robust implantable sensors.

JTD Keywords: Biomedicine, Electrochemistry, Endoscope, Implantable device, Ion-selective electrode (ISE) sensor, Ischemia, pH detection, Biocompatibility, Chemical sensors, Electrochemistry, Electrodes, Electropolymerization, Endoscopy, Functional polymers, Implants (surgical), Ion selective electrodes, Medical applications, Polyvinyl chlorides, Stabilization, Biomedical applications, Biomedicine, Implantable devices, Ion selective sensors, Ischemia, Membrane instability, pH detection, Poly(3 ,4 ethylenedioxythiophene) (PEDOT), Ion selective membranes

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro-and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules-DNA or proteins-to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.

JTD Keywords: Atomic force microscopy, Cations, Force spectroscopy, Lipid bilayer, Mechanical stability

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.

JTD Keywords: Chain-Length, Spectroscopy, Nanomechanics, Microscopy, Elasticity, Stability, Membranes, Reveals, Fusion, Ions

Proteins are fundamental molecules in biology that are also involved in a wide range of industrial and biotechnological processes. Consequently, many works in the literature have been devoted to the study of protein-protein and protein-surface interactions in aqueous solutions. The results have been usually interpreted within the frame of the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory for colloidal systems. However, against the DLVO predictions, striking evidence of repulsive forces between proteins at high salt concentrations has been observed in different works based on the analysis of the second virial coefficient or on the direct measurement of protein interaction with an atomic force microscope. Hydration forces due to the adsorption of hydrated cations onto the negatively charged protein surfaces have been invoked to rationalize this anomalous repulsion. The hydration forces between proteins provide protein-covered particles with a non-DLVO colloidal stability at high salt concentrations, as different studies in the literature has proven. This review summarizes the most relevant results published so far on the presence of hydration forces between proteins and protein-coated colloidal particles.

JTD Keywords: Colloidal particles, Colloidal stability, Hydrated ions, Hydration forces, Proteins

An important goal in cluster analysis is the internal validation of results using an objective criterion. Of particular relevance in this respect is the estimation of the optimum number of clusters capturing the intrinsic structure of your data. This paper proposes a method to determine this optimum number based on the evaluation of fuzzy partition stability under bootstrap resampling. The method is first characterized on synthetic data with respect to hyper-parameters, like the fuzzifier, and spatial clustering parameters, such as feature space dimensionality, clusters degree of overlap, and number of clusters. The method is then validated on experimental datasets. Furthermore, the performance of the proposed method is compared to that obtained using a number of traditional fuzzy validity rules based on the cluster compactness-to-separation criteria. The proposed method provides accurate and reliable results, and offers better generalization capabilities than the classical approaches.

JTD Keywords: Fuzzy c-means, Cluster validity, Number of clusters, Cluster stability

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein similar to 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

JTD Keywords: Soft lithography, Cell-adhesion, Microstructures, Fabrication, Stability, Patterns

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JTD Keywords: Self-assembled monolayers, High-aspect-ratio, Dip-pen nanolithography, Langmuir-blodgett-films, Conducting polymer, Enzymes, Microstructures, Immunosensors, Fabrication, Stability