Structure-Dependent Water Responsiveness of Protein Block Copolymers.

Biological water-responsive (WR) materials are abundant in nature, and they are used as mechanical actuators for seed dispersal by many plants such as wheat awns and pinecones. WR biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural water evaporation. Recent work on WR silk proteins has shown that ß-sheet nanocrystalline domains with high stiffness correlate with the high WR actuation energy density, but the fundamental mechanisms to drive water responsiveness in proteins remain poorly understood. Here, we design, synthesize, and study protein block copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E), namely, CEC. We use these protein materials to create WR actuators with energy densities that outperform mammalian muscle. To elucidate the effect of structure on WR actuation, CEC was compared to a variant, CECL44A, in which a point mutation disrupts the α-helical structure of the C domain. Surprisingly, CECL44A outperformed CEC, showing higher energy density and less susceptibility to degradation after repeated cycling. We show that CECL44A exhibits a higher degree of intermolecular interactions and is stiffer than CEC at high relative humidity (RH), allowing for less energy dissipation during water responsiveness. These results suggest that strong intermolecular interactions and the resulting, relatively steady protein structure are important for water responsiveness.

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment.

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

Enhanced water-responsive actuation of porous Bombyx mori silk.

Bombyx mori silk with a nanoscale porous architecture significantly deforms in response to changes in relative humidity. Despite the increasing amount of water adsorption and water-responsive strain with increasing porosity of the silk, there is a range of porosities that result in silk's optimal water-responsive energy density at 3.1 MJ m-3. Our findings show the possibility of controlling water-responsive materials' swelling pressure by controlling their nanoporosities.

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

Traumatic temporal bone fracture with middle ear effusion: A case report.

Temporal bone fracture is a relatively rare but significant complication of traumatic head injury. We present a rare and unique case of traumatic temporal bone fracture with middle ear effusion, in a 76-year-old woman, following a fall. Physical examination on presentation was remarkable for a superficial scalp hematoma in the occipital region, without any focal neurological deficits. An initial non-contrast head CT revealed a large posterior scalp hematoma and subtle changes suggestive of artifact vs. hemorrhage within the right temporal lobe. Over two days, she developed a worsening headache, with new hearing impairment and reduced right sided bone-conduction on auditory testing. A repeat head CT confirmed a right hemorrhagic temporal lobe contusion as well as a right mastoid and middle ear effusion. A dedicated temporal bone CT scan was performed, which revealed an acute longitudinal fracture through the right mastoid bone without extension into the middle ear cavity. She was evaluated by neurosurgery, managed symptomatically, and observed closely. Her neurological status remained stable, and she was discharged with planned outpatient follow-up with her primary care provider and the consulting neurosurgeon. This case illustrates sequalae of traumatic temporal bone fracture, as well as the value of clinical history and heightened clinical concern for an occult, easily overlooked region during imaging.

Tuning water-responsiveness with Bombyx mori silk-silica nanoparticle composites.

Bombyx (B.) mori silk's water-responsive actuation correlates to its high ß-sheet crystallinity. In this research, we demonstrated that stiff silica nanoparticles can mimic the role of dispersed ß-sheet nanocrystals and dramatically increase amorphous silk's water-responsive actuation energy density to ∼700 kJ m-3.

No ordinary proteins: Adsorption and molecular orientation of monoclonal antibodies.

The interaction of monoclonal antibodies (mAbs) with air/water interfaces plays a crucial role in their overall stability in solution. We aim to understand this behavior using pendant bubble measurements to track the dynamic tension reduction and x-ray reflectivity to obtain the electron density profiles (EDPs) at the surface. Native immunoglobulin G mAb is a rigid molecule with a flat, "Y" shape, and simulated EDPs are obtained by rotating a homology construct at the surface. Comparing simulations with experimental EDPs, we obtain surface orientation probability maps showing mAbs transition from flat-on Y-shape configurations to side-on or end-on configurations with increasing concentration. The modeling also shows the presence of ß sheets at the surface. Overall, the experiments and the homology modeling elucidate the orientational phase space during different stages of adsorption of mAbs at the air/water interface. These finding will help define new strategies for the manufacture and storage of antibody-based therapeutics.

Design Strategies to Tune the Structural and Mechanical Properties of Synthetic Collagen Hydrogels.

As an important component of biomaterials, collagen provides three-dimensional scaffolds and biological cues for cell adhesion and proliferation in tissue engineering. Recombinant collagen-like proteins, which were initially discovered in Streptococcus pyogenes and produced in heterologous hosts, have been chemically and genetically engineered for biomaterial applications. However, existing collagen-like proteins do not form gels, limiting their utility as biomaterials. Here, we present a series of rationally designed collagen-like proteins composed of a trimerization domain, triple-helical domains with various lengths, and a pair of heterotrimeric coiled-coil sequences attached to the N- and C-termini as adhesive ends. These designed proteins fold into triple helices and form self-supporting gels. As the triple-helical domains are lengthened, the gels become less stiff, pore sizes increase, and structural anisotropy decreases. Moreover, cell-culture assay confirms that the designed proteins are noncytotoxic. This study provides a design strategy for collagen-based biomaterials. The sequence variations reveal a relationship between the protein primary structure and material properties, where variations in the cross-linking density and association energies define the gelation of the protein network.

Environmental health conditions in the transitional stage of forcible displacement: A systematic scoping review.

In 2019, 30,000 people were forced to leave their homes due to conflict, persecution, and natural disaster each day. Eighty-five percent of refugees live in developing countries, and they often face underfunded and inadequate environmental health services. Many displaced persons live in camps and other temporary settlements long after the displacement event occurs. However, there is little evidence on environmental health conditions in the transitional phase-defined by the United Nations High Commissioner for Refugees as six months to two years after displacement. To address this gap in research, we conducted a systematic scoping review of environmental health conditions, exposures, and outcomes in transitional displacement settings, as well as reported obstacles and recommendations for improvement. Eighty-eight publications met the inclusion criteria. Water supply was the most frequently discussed environmental health topic. Overcrowding was the most common risk factor reported, Vibrio cholerae was the most common pathogen reported, and diarrhea was the most commonly reported health outcome. Obstacles and recommendations were categorized as institutional, political or implementation-based. Identified knowledge gaps included minimal information on setting logistics and on topics such as menstrual hygiene, oral hygiene and fomite contamination. In order to improve environmental health conditions in transitional displacement settings, all levels of government and non-governmental organizations should increase collaboration to improve resource provision. This study is the first to report on environmental health conditions in this important time of transition between the emergency and protracted stages of displacement.

Environmental health conditions in protracted displacement: A systematic scoping review.

Adequate environmental health services are critical for human rights, health, and development, especially in the context of forced displacement. There are more than 70 million forcibly displaced persons worldwide, most in protracted situations, having been displaced for more than two years. Some live in camps or informal settlements, but most live in urban areas. Environmental health services are important in the transition from emergency response to sustainable development in these settings, but evidence on environmental health in displaced populations is disparate and of variable quality. We conducted a systematic scoping review of environmental conditions, exposures, and outcomes in protracted displacement settings; obstacles to improvement in environmental health services; and recommendations made for improvement. We included 213 publications from peer-reviewed and grey literature databases. Data were extracted on environmental health topics including water, sanitation, hygiene, overcrowding, waste management, energy supply, vector control, menstrual hygiene, air quality, and food safety. Most studies present data from low- and lower-middle income countries. Northern Africa and Western Asia and Sub-Saharan Africa are the most-represented regions. There is substantial evidence on water, sanitation, and crowding, but few studies report findings on other environmental health topics. Water-related disease, parasites, and respiratory infections are frequently cited and studies report that services often fail to meet international standards for humanitarian response. The most frequent obstacles and recommendations are institutional, political, or implementation-related, but few studies provide concrete recommendations for improvement. Our review compiles and characterizes the research on environmental health in protracted displacement. We recommend including displaced populations in international environmental health policy and monitoring initiatives, and bridging from humanitarian response to sustainable development by preparing for long-term displacement from the early stages of a crisis.

ß-Sheet Nanocrystals Dictate Water Responsiveness of Bombyx Mori Silk.

Water-responsive (WR) materials that strongly swell and shrink in response to changes in relative humidity (RH) have shown a great potential to serve as high-energy actuators for soft robotics and new energy-harvesting systems. However, the design criteria governing the scalable and high-efficiency WR actuation remain unclear, and thus inhibit further development of WR materials for practical applications. Nature has provided excellent examples of WR materials that contain stiff nanocrystalline structures that can be crucial to understand the fundamentals of WR behavior. This work reports that regenerated Bombyx (B.) mori silk can be processed to increase ß-sheet crystallinity, which dramatically increases the WR energy density to 1.6 MJ m-3 , surpassing that of all known natural muscles, including mammalian muscles and insect muscles. Interestingly, the maximum water sorption decreases from 80.4% to 19.2% as the silk's ß-sheet crystallinity increases from 19.7% to 57.6%, but the silk's WR energy density shows an eightfold increase with higher fractions of ß-sheets. The findings of this study suggest that high crystallinity of silk reduces energy dissipation and translates the chemical potential of water-induced pressure to external loads more efficiently during the hydration/dehydration processes. Moreover, the availability of B. mori silk opens up possibilities for simple and scalable modification and production of powerful WR actuators.

Armoring the Interface with Surfactants to Prevent the Adsorption of Monoclonal Antibodies.

The pharmaceutical industry uses surface-active agents (excipients) in protein drug formulations to prevent the aggregation, denaturation, and unwanted immunological response of therapeutic drugs in solution as well as at the air/water interface. However, the mechanism of adsorption, desorption, and aggregation of proteins at the interface in the presence of excipients remains poorly understood. The objective of this work is to explore the molecular-scale competitive adsorption process between surfactant-based excipients and two monoclonal antibody (mAb) proteins, mAb-1 and mAb-2. We use pendant bubble tensiometry to measure the ensemble average adsorption dynamics of mAbs with and without the excipient. The surface tension measurements allow us to quantify the rate at which the molecules "race" to the interface in single-component and mixed systems. These results define the phase space, where coadsorption of both mAbs and excipients occurs onto the air/water interface. In parallel, we use X-ray reflectivity (XR) measurements to understand the molecular-scale dynamics of competitive adsorption, revealing the surface-adsorbed amounts of the antibody and excipient. XR has revealed that at a sufficiently high surface concentration of the excipient, mAb adsorption to the surface and subsurface domains was inhibited. In addition, despite the fact that both mAbs adsorb via a similar mechanistic pathway and with similar dynamics, a key finding is that the competition for the interface directly correlates with the surface activity of the two mAbs, resulting in a fivefold difference in the concentration of the excipient needed to displace the antibody.

Environmental health in forced displacement: A systematic scoping review of the emergency phase.

There are 70.8 million forcibly displaced people worldwide, including internally displaced persons, refugees, and asylum seekers. Since mortality rates are highest in the first six months of displacement, the provision of adequate services and infrastructure by relief organizations is critical in this "emergency phase." Environmental health provisions such as adequate water supply, excreta management, solid waste management, and vector control measures are among those essential services. We conducted a systematic scoping review of environmental health in the emergency phase of displacement (the six months following first displacement). A total of 122 publications, comprising 104 peer-reviewed and 18 grey literature publications, met the inclusion criteria. We extracted data relating to environmental health conditions and services, associated outcomes, and information concerning obstacles and recommendations for improving these conditions and services. Despite the fact that most displaced people live outside of camps, publications largely report findings for camps (n = 73, 60%). Water supply (n = 57, 47%) and excreta management (n = 47, 39%) dominate the literature. Energy access (n = 7, 6%), exposure to harsh weather from inadequate shelter (n = 5, 4%), food hygiene and safety (n = 4, 3%), indoor air quality (n = 3, 3%), menstrual hygiene management (n = 2, 2%), dental hygiene (n = 2, 2%), and ambient air quality (n = 1, 1%) are relatively understudied. The most common health outcome attributed to inadequate environmental conditions in the included publications is diarrhea (n = 43, 35%). We found that organizations and governments often embrace their own standards, however we call for policymakers to adopt standards no less rigorous than Sphere for the emergency phase of displacement. Although other reviews examine water, sanitation, and hygiene interventions in emergencies, this is the first systematic review of environmental health more broadly in the first six months of displacement.

Effect of Orientation and Wetting Properties on the Behavior of Janus Particles at the Air-Water Interface.

The adhesion force and contact angle of gold-capped silica Janus particles and plain silica particles at an air-water interface are studied via colloidal atomic force microscopy. Particles are attached to cantilevers at various orientations, and wetting properties of the gold surface are varied through modification with dodecanethiol. Thiol modification increases the hydrophobicity of the gold surface, thereby increasing the difference between the contact angles of the gold hemisphere and the silica hemisphere and, thus, increasing the degree of amphiphilicity of the Janus particle. Subsequently, the colloidal probe is pushed into a stationary bubble from the water phase followed by retraction back into the water phase. Adhesion force is found to be higher for Janus particles than isotropic silica particles, regardless of orientation of the anisotropic hemisphere. Particles with their polar half oriented toward the water and apolar half facing the air show an increase in adhesion force and contact angle as the degree of amphiphilicity of the particles increases. For particles of the reverse orientation, no significant difference is observed as wetting properties change. Both adhesion force and contact angle display an inverse relationship with a cap angle for particles with a higher degree of amphiphilicity. These results are of importance for using Janus particles to stabilize interfaces as well as for understanding the equilibrium height of Janus particles at the interface, which will impact capillary interactions and thus self-assembly.

Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems.

Impact of Surface Amphiphilicity on the Interfacial Behavior of Janus Particle Layers under Compression.

Langmuir monolayers of silica/gold Janus particles with two different degrees of amphiphilicity have been examined to study the significance of particle surface amphiphilicity on the structure and mechanical properties of the interfacial layers. The response of the layers to the applied compression provides insight into the nature and strength of the interparticle interactions. Different collapse modes observed for the interfacial layers are linked to the amphiphilicity of Janus particles and their configuration at the interface. Molecular dynamics simulations on nanoparticles with similar contact angles provide insight on the arrangement of particles at the interface and support our conclusion that the interfacial configuration and collapse of anisotropic particles at the air/water interface are controlled by particle amphiphilicity.

Incentivizing Physician Diversity in Radiology.

In this article, the authors review the evolving state of diversity in the field of radiology. The authors discuss several early and recent historical legislative milestones that increased the equitable delivery of health care in the United States, such as Title VI of the Civil Rights Act of 1964, which ensured that funds for Medicare reimbursement would be available only to desegregated hospitals. Furthermore, the authors examine the current state of diversity and representation in radiology, in which underrepresented minorities represent 8.3% of training and practicing radiologists, and women represent 27.8% of radiologists. Finally, the authors present arguments for diversity in the current medical education system, analyze hurdles to increasing representation in radiology, and consider the future of diversity and inclusiveness in the field.

Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective.

Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor-liquid, solid-liquid, and liquid-liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended.

'Reverse' Hofmeister effects on the sol-gel transition rates for an α-helical peptide-PEG bioconjugate.

We examine the dynamics of the sol-gel transition for end-functionalized linear- and 4-arm-peptides bioconjugated to poly-ethylene glycol (PEG) in aqueous environments with increasingly chaotropic (Cl- < Br- < I-) anions. A 23-amino acid peptide sequence is rationally designed to self-assemble upon folding into the ordered α-helical conformation due to the hydrophobic effect. We use Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) to quantify the ensemble average reversible secondary structure transitions as a function of electrolyte concentration and specific ion effects along the Hofmeister series. Subsequently, microrheology is used to quantify the kinetics of the gelation process, as it relates to folding and specific ion interactions. Our key findings were non-intuitive. We observe the faster evolution of the gel transitions in systems with more chaotropic anions. For our peptides in aqueous solution, "water-structuring" ions yield faster assembly behavior with a viscoelastic exponent, n, closer to unity representing self-assemblies that are Rouse-like. In contrast, ions that are "water-breaking" resulted in smaller viscoelastic exponents where self-assembly dynamics result in a viscoelastic exponent that suggests polymer entanglements.