Polypeptide organic radical batteries.

In only a few decades, lithium-ion batteries have revolutionized technologies, enabling the proliferation of portable devices and electric vehicles1, with substantial benefits for society. However, the rapid growth in technology has highlighted the ethical and environmental challenges of mining lithium, cobalt and other mineral ore resources, and the issues associated with the safe usage and non-hazardous disposal of batteries2. Only a small fraction of lithium-ion batteries are recycled, further exacerbating global material supply of strategic elements3-5. A potential alternative is to use organic-based redox-active materials6-8 to develop rechargeable batteries that originate from ethically sourced, sustainable materials and enable on-demand deconstruction and reconstruction. Making such batteries is challenging because the active materials must be stable during operation but degradable at end of life. Further, the degradation products should be either environmentally benign or recyclable for reconstruction into a new battery. Here we demonstrate a metal-free, polypeptide-based battery, in which viologens and nitroxide radicals are incorporated as redox-active groups along polypeptide backbones to function as anode and cathode materials, respectively. These redox-active polypeptides perform as active materials that are stable during battery operation and subsequently degrade on demand in acidic conditions to generate amino acids, other building blocks and degradation products. Such a polypeptide-based battery is a first step to addressing the need for alternative chemistries for green and sustainable batteries in a future circular economy.

Analysis of Large Data Sets in a Physical Chemistry Laboratory NMR Experiment Using Python.

We describe an update to an experiment demonstrating low-field NMR spectroscopy in the undergraduate physical chemistry laboratory. A Python-based data processing and analysis protocol is developed for this experiment. The Python language is used in fillable worksheets in the notebook software JupyterLab, providing an interactive means for students to work with the measured data step by step. The protocol teaches methods for the analysis of large data sets in science or engineering, a topic that is absent from traditional chemistry curricula. Python is among the most widely used modern tools for data analysis. In addition, its open-source nature reduces the barriers for adoption in an educational laboratory.

Tensile force-induced cytoskeletal remodeling: Mechanics before chemistry.

Understanding cellular remodeling in response to mechanical stimuli is a critical step in elucidating mechanical activation of biochemical signaling pathways. Experimental evidence indicates that external stress-induced subcellular adaptation is accomplished through dynamic cytoskeletal reorganization. To study the interactions between subcellular structures involved in transducing mechanical signals, we combined experimental data and computational simulations to evaluate real-time mechanical adaptation of the actin cytoskeletal network. Actin cytoskeleton was imaged at the same time as an external tensile force was applied to live vascular smooth muscle cells using a fibronectin-functionalized atomic force microscope probe. Moreover, we performed computational simulations of active cytoskeletal networks under an external tensile force. The experimental data and simulation results suggest that mechanical structural adaptation occurs before chemical adaptation during filament bundle formation: actin filaments first align in the direction of the external force by initializing anisotropic filament orientations, then the chemical evolution of the network follows the anisotropic structures to further develop the bundle-like geometry. Our findings present an alternative two-step explanation for the formation of actin bundles due to mechanical stimulation and provide new insights into the mechanism of mechanotransduction.

Effects of Glutathione and Histidine on NO Release from a Dimeric Dinitrosyl Iron Complex (DNIC).

Rates of NO release from synthetic dinitrosyl iron complexes (DNICs) are shown to be responsive to coordination environments about iron. The effect of biologically relevant cellular components, glutathione and histidine, on the rate of NO release from a dimeric, "Roussin's Red Ester", DNIC with bridging µ-S thioglucose ligands, SGlucRRE or [(µ-SGluc)Fe(NO)2]2 (SGluc = 1-thio-ß-d-glucose tetraacetate), was investigated. From the Griess assay and X-band EPR data, decomposition of the product from the histidine-cleaved dimer, [(SGluc)(NHis)Fe(NO)2], generated Fe(III) and increased the NO release rate in aqueous media when compared to the intact SGlucRRE precursor. In contrast, increasing concentrations of exogenous glutathione generated the stable [(SGluc)(GS)Fe(NO)2]- anion and depressed the rate of NO release. Both of the cleaved, monomeric intermediates were characterized with ESI-MS, EPR, and FT-IR spectroscopies. On the basis of the Griess assay coupled with data from an intracellular fluorometric probe, both the monomeric DNICs and dimeric SGlucRRE diffuse into smooth muscle cells, chosen as appropriate archetypes of vascular relaxation, and release their NO payload. Ultimately, this work provides insight into tuning NO release beyond the design of DNICs, through the incubation with safe, accessible biological molecules.

Toward the Optimization of Dinitrosyl Iron Complexes as Therapeutics for Smooth Muscle Cells.

In this study, dinitrosyl iron complexes (DNICs) are shown to deliver nitric oxide (NO) into the cytosol of vascular smooth muscle cells (SMCs), which play a major role in vascular relaxation and contraction. Malfunction of SMCs can lead to hypertension, asthma, and erectile dysfunction, among other disorders. For comparison of the five DNIC derivatives, the following protocols were examined: (a) the Griess assay to detect nitrite (derived from NO conversion) in the absence and presence of SMCs; (b) the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay for cell viability; (c) an immunotoxicity assay to establish if DNICs stimulate immune response; and (d) a fluorometric assay to detect intracellular NO from treatment with DNICs. Dimeric Roussin's red ester (RRE)-type {Fe(NO)2}9 complexes containing phenylthiolate bridges, [(µ-SPh)Fe(NO)2]2 or SPhRRE, were found to deliver NO with the lowest effect on cell toxicity (i.e., highest IC50). In contrast, the RRE-DNIC with the biocompatible thioglucose moiety, [(µ-SGlu)Fe(NO)2]2 (SGlu = 1-thio-ß-d-glucose tetraacetate) or SGluRRE, delivered a higher concentration of NO to the cytosol of SMCs with a 10-fold decrease in IC50. Additionally, monomeric DNICs stabilized by a bulky N-heterocyclic carbene (NHC), namely, 1,3-bis(2,4,6-trimethylphenyl)imidazolidene (IMes), were synthesized and yielded the DNIC complexes SGluNHC, [IMes(SGlu)Fe(NO)2], and SPhNHC, [IMes(SPh)Fe(NO)2]. These oxidized {Fe(NO)2}9 NHC DNICs have an IC50 of ∼7 µM; however, the NHC-based complexes did not transfer NO into the SMC. Per contra, the reduced, mononuclear {Fe(NO)2}10 neocuproine-based DNIC, neoDNIC, depressed the viability of the SMCs, as well as generated an increase of intracellular NO. Regardless of the coordination environment or oxidation state, all DNICs showed a dinitrosyl iron unit (DNIU)-dependent increase in viability. This study demonstrates a structure-function relationship between the DNIU coordination environment and the efficacy of the DNIC treatments.

Harnessing the Chemical Diversity of the Natural Product Magnolol for the Synthesis of Renewable, Degradable Neolignan Thermosets with Tunable Thermomechanical Characteristics and Antioxidant Activity.

Magnolol, a neolignan natural product with antioxidant properties, contains inherent, orthogonal, phenolic, and alkenyl reactive groups that were used in both direct thermoset synthesis, as well as the stepwise synthesis of a small library of monomers, followed by transformation into thermoset materials. Each monomer from the small library was prepared via a single step functionalization reaction of the phenolic groups of magnolol. Thermoset materials were realized through solvent-free, thiol-ene reactions, and the resulting cross-linked materials were each comprised of thioether and ester linkages, with one retaining the hydrophilic phenols from magnolol, another having the phenols protected as an acetonide, and two others incorporating the phenols into additional cross-linking sites via hydrolytically labile carbonates or stable ether linkages. With this diversity of chemical compositions and structures, the thermosets displayed a range of thermomechanical properties including glass transition temperatures, Tg, 29-52 °C, onset of thermal degradation, Td, from about 290-360 °C, and ultimate strength up to 50 MPa. These tunable materials were studied in their degradation and biological properties with the aim of exploiting the antioxidant properties of the natural product. Hydrolytic degradation occurred under basic conditions (pH = 11) in all thermosets, but with kinetics that were dependent upon their chemical structures and mechanical properties: 20% mass loss was observed at 5, 7, 27, and 40 weeks for the thermosets produced from magnolol directly, acetonide-protected magnolol, bis(allyl carbonate)-functionalized magnolol, and bis(allyl ether)-functionalized magnolol, respectively. Isolated degradation products and model compounds displayed antioxidant properties similar to magnolol, as determined by both UV-vis and in vitro reactive oxygen species (ROS) assays. As these magnolol-based thermosets were found to also allow for extended cell culture, these materials may serve as promising degradable biomaterials.

Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-ß.

Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2(-/-) mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-ß). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-ß activation and Acta2(-/-) SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-ß, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations.

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells.

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5h, while a significant inhibition was recorded at 24h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.

Specific anion effects on water structure adjacent to protein monolayers.

Vibrational sum frequency spectroscopy (VSFS) was used to explore specific ion effects on interfacial water structure adjacent to a bovine serum albumin (BSA) monolayer adsorbed at the air/water interface. The subphase conditions were varied by the use of six different sodium salts and four different pH values. At pH 2 and 3, the protein layer was positively charged and it was found that the most chaotropic anions caused the greatest attenuation of water structure. The order of the salts followed an inverse Hofmeister series. On the other hand, when the protein layer was near its isoelectric point (pH 5), the most chaotropic anions caused the greatest increase in water structure, although the effect was weak. In this case, a direct Hofmeister series was obeyed. Finally, virtually no effect was observed when the protein layer was negatively charged with a subphase pH of 9. For comparison, similar experiments were run with positively charged, negatively charged, and zwitterionic surfactant monolayers. These experiments gave rise to nearly the identical results as the protein monolayer which suggested that specific anion effects are dominated by the charge state of the interfacial layer rather than its detailed chemical structure. In a final set of experiments, salt effects were examined with a monolayer made from an elastin-like polypeptide (ELP). The peptide consisted of 120 pentameric repeats of the sequence Val-Pro-Gly-Val-Gly. Data from this net neutral biopolymer followed a very weak, but direct Hofmeister series. This suggested that direct anion binding to the amide groups in the backbone of a polypeptide is quite weak in agreement with the BSA data. The results from the variously charged protein, surfactant, and polymer monolayers were compared with a modified Gouy-Chapman-Stern model. The agreement with this simple model was quite good.

Integrated microscopy for real-time imaging of mechanotransduction studies in live cells.

Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.

Toward biocompatible dinitrosyl iron complexes: sugar-appended thiolates.

Both monomeric and dimeric tetraacetylglucose-containing {Fe(NO)2}9 dinitrosyl iron complexes (DNICs) were prepared and examined for NO release in the presence of both chemical NO-trapping agents and endothelial cells.

Selective regulation of cytoskeletal tension and cell-matrix adhesion by RhoA and Src.

The crosstalk between cells and their microenvironment enables cellular adaptation to external mechanical cues through the remodeling of cytoskeletal structures and cell-matrix adhesions to ensure normal cell function. This study investigates the relationship between the cytoskeletal tension and integrin α5ß1 adhesion strength to the matrix (i.e. fibronectin) in the context of RhoA-Src crosstalk. Integration of atomic force microscopy (AFM) with total internal reflection fluorescence and spinning-disk confocal microscopy enabled acquisition of complementary structural and functional measurements on live vascular smooth muscle cells expressing RhoA and c-Src variants (wild-type, dominant negative, constitutively active). Single ligand-receptor interaction measurements performed with AFM probes functionalized with fibronectin showed that RhoA and c-Src activation have different effects on cytoskeletal tension development, inducing two distinct force-stiffness functional regimes for α5ß1-integrin binding to fibronectin. Moreover, fluorescence measurements showed that c-Src activation had a modest effect on actin morphology, while RhoA significantly modulated stress fiber formation. In addition, c-Src was associated with regulation of myosin light chain (MLC) phosphorylation, suggesting a c-Src-dependent modulation of RhoA pathway through activation of downstream effectors. Therefore, c-Src may be a possible component of cytoskeletal tension regulation through MLC activation. Our findings suggest that Src and RhoA coordinate a regulatory network that determines cytoskeletal tension through activation of actomyosin contractility. In turn, the cytoskeletal tension state modulates integrin α5ß1-fibronectin adhesion force.

RhoA-induced cytoskeletal tension controls adaptive cellular remodeling to mechanical signaling.

The ability to measure real-time mechanosensitive events at the subcellular level in response to discrete mechanical stimulation is a critical component in understanding mechanically-induced cellular remodeling. Vascular smooth muscle cells (VSMC) were transfected with RhoA constructs (wild type, dominant negative or constitutively active) or treated with ML-7 to induce specific cytoskeletal tension characteristics prior to mechanical stimulation. Tensile stress was applied to live VSMC using an atomic force microscope probe functionalized with extracellular matrix (ECM) proteins. The ECM induces selective integrin activation and focal adhesion formation, enabling direct manipulation of cortical actin through an active ECM-integrin-actin linkage. Therefore, locally induced mechanosensitive events triggered downstream activation of intracellular signaling pathways responsible for actin and focal adhesion remodeling throughout the cell. Integration of mechanical stimulation with simultaneous fluorescence imaging by spinning-disk confocal and total internal reflection fluorescence microscopy enabled visualization and quantification of molecular dynamic events at the sub-cellular level in real-time. Results provide evidence that the pre-existing cytoskeletal tension affects the actomyosin apparatus which in turn coordinates the ability of the cell to adapt to the externally applied stress. RhoA activation induced high cytoskeletal tension that correlated with increased stress fiber formation, cell stiffness, integrin activation and myosin phosphorylation. In contrast, blocking Rho-kinase or myosin function was characterized by low cytoskeletal tension with a decreased level of stress fiber formation, lower cell stiffness and integrin activation. Our findings show that VSMC sense and adapt to physical microenvironmental changes by a coordinated response of the actomyosin apparatus necessary to establish a new homeostatic state.

Live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

On the mechanism of the hofmeister effect.

Vibrational sum frequency spectroscopy was used to probe fatty amine monolayers spread on various electrolyte solutions. The spectra revealed ion specific changes in both monolayer ordering and water structure with the former following the Hofmeister series. Separate measurements of the surface potential as a function of ion tracked closely to changes in alkyl chain structure, but less closely to changes in water structure. The disruption of the monolayer ordering could be ascribed to the relative ability of the ions to penetrate past the hydrophilic surface of the monolayer's headgroups and into the more hydrophobic portion of the thin film. The corresponding trends observed in the surface water structure showed significant deviations from the Hofmeister series, leading to the conclusion that the changes in surface water structure, often credited with being the origin of Hofmeister effects, are probably not of primary importance. On the other hand, dispersion forces almost certainly play a large role in the order of the Hofmeister series.

The Vroman effect: a molecular level description of fibrinogen displacement.

The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged alpha C domains on HPF sit between the rest of the macromolecule and the underlying surface. The alpha C domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the alpha C domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.

Thermodynamics of phase transitions in langmuir monolayers observed by vibrational sum frequency spectroscopy.

Vibrational sum-frequency spectroscopy (VSFS) was used to study gauche defects in octadecylamine (ODA) monolayers at the air/water interface. The VSFS spectra provide unique insights into phase transitions that occur as a result of changes in the structure of the monolayer's hydrophobic region. These changes can be attributed to the increased presence of gauche conformers in the ODA alkyl chains during the monolayer's transition from the solid to liquid phase. Temperature-dependent spectra from monolayers at several different pressures were used to assign the phase transition temperature based on the observed changes in microscopic structure. Through application of a two-dimensional form of the Clapeyron equation, the first in situ measurements of the entropy and enthalpy changes associated with gauche conformers in a monolayer were made.