Expression Microdissection for the Analysis of miRNA in a Single-Cell Type.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

Enhancing Crystallization in Hybrid Perovskite Solar Cells Using Thermally Conductive 2D Boron Nitride Nanosheet Additive.

Controlling crystallization and grain growth is crucial for realizing highly efficient hybrid perovskite solar cells (PSCs). In this work, enhanced PSC photovoltaic performance and stability by accelerating perovskite crystallization and grain growth via 2D hexagonal boron nitride (hBN) nanosheet additives incorporated into the active perovskite layer are demonstrated. In situ X-ray scattering and infrared thermal imaging during the perovskite annealing process revealed the highly thermally conductive hBN nanosheets promoted the phase conversion and grain growth in the perovskite layer by facilitating a more rapid and spatially uniform temperature rise within the perovskite film. Complementary structural, physicochemical, and electrical characterizations further showed that the hBN nanosheets formed a physical barrier at the perovskite grain boundaries and the interfaces with charge transport layers, passivating defects, and retarding ion migration. As a result, the power conversion efficiency of the PSC is improved from 17.4% to 19.8%, along with enhanced device stability, retaining ≈90% of the initial efficiency even after 500 h ambient air storage. The results not only highlight 2D hBN as an effective additive for PSCs but also suggest enhanced thermal transport as one of the pathways for improved PSC performance by 2D material additives in general.

Recent advances of two-dimensional material additives in hybrid perovskite solar cells.

Perovskite solar cells (PSCs) have become one of the state-of-the-art photovoltaic technologies due to their facile solution-based fabrication processes combined with extremely high photovoltaic performance originating from excellent optoelectronic properties such as strong light absorption, high charge mobility, long free charge carrier diffusion length, and tunable direct bandgap. However, the poor intrinsic stability of hybrid perovskites under environmental stresses including light, heat, and moisture, which is often associated with high defect density in the perovskite, has limited the large-scale commercialization and deployment of PSCs. The use of process additives, which can be included in various subcomponent layers in the PSC, has been identified as one of the effective approaches that can address these issues and improve the photovoltaic performance. Among various additives that have been explored, two-dimensional (2D) materials have emerged recently due to their unique structures and properties that can enhance the photovoltaic performance and device stability by improving perovskite crystallization, defect passivation, and charge transport. Here, we provide a review of the recent progresses in 2D material additives for improving the PSC performance based on key representative 2D material systems, including graphene and its derivatives, transitional metal dichalcogenides, and black phosphorous, providing a useful guideline for further exploiting unique nanomaterial additives for more efficient and stable PSCs in the near future.

Molecular Basis for Surface-Initiated Non-Thrombin-Generated Clot Formation Following Viral Infection.

In this paper, we propose a model that connects two standard inflammatory responses to viral infection, namely, elevation of fibrinogen and the lipid drop shower, to the initiation of non-thrombin-generated clot formation. In order to understand the molecular basis for the formation of non-thrombin-generated clots following viral infection, human epithelial and Madin-Darby Canine Kidney (MDCK, epithelial) cells were infected with H1N1, OC43, and adenovirus, and conditioned media was collected, which was later used to treat human umbilical vein endothelial cells and human lung microvascular endothelial cells. After direct infection or after exposure to conditioned media from infected cells, tissue surfaces of both epithelial and endothelial cells, exposed to 8 mg/mL fibrinogen, were observed to initiate fibrillogenesis in the absence of thrombin. No fibers were observed after direct viral exposure of the endothelium or when the epithelium cells were exposed to SARS-CoV-2 isolated spike proteins. Heating the conditioned media to 60 °C had no effect on fibrillogenesis, indicating that the effect was not enzymatic but rather associated with relatively thermally stable inflammatory factors released soon after viral infection. Spontaneous fibrillogenesis had previously been reported and interpreted as being due to the release of the alpha C domains due to strong interactions of the interior of the fibrinogen molecule in contact with hydrophobic material surfaces rather than cleavage of the fibrinopeptides. Contact angle goniometry and immunohistochemistry were used to demonstrate that the lipids produced within the epithelium and released in the conditioned media, probably after the death of infected epithelial cells, formed a hydrophobic residue responsible for fibrillogenesis. Hence, the standard inflammatory response constitutes the ideal conditions for surface-initiated clot formation.

Control of anti-thrombogenic properties: surface-induced self-assembly of fibrinogen fibers.

Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.

Engineering of bio-hybrid materials by electrospinning polymer-microbe fibers.

Although microbes have been used in industrial and niche applications for several decades, successful immobilization of microbes while maintaining their usefulness for any desired application has been elusive. Such a functionally bioactive system has distinct advantages over conventional batch and continuous-flow microbial reactor systems that are used in various biotechnological processes. This article describes the use of polyethylene oxide(99)-polypropylene oxide(67)-polyethylene oxide(99) triblock polymer fibers, created via electrospinning, to encapsulate microbes of 3 industrially relevant genera, namely, Pseudomonas, Zymomonas, and Escherichia. The presence of bacteria inside the fibers was confirmed by fluorescence microscopy and SEM. Although the electrospinning process typically uses harsh organic solvents and extreme conditions that generally are harmful to bacteria, we describe techniques that overcome these limitations. The encapsulated microbes were viable for several months, and their metabolic activity was not affected by immobilization; thus they could be used in various applications. Furthermore, we have engineered a microbe-encapsulated cross-linked fibrous polymeric material that is insoluble. Also, the microbe-encapsulated active matrix permits efficient exchange of nutrients and metabolic products between the microorganism and the environment. The present results demonstrate the potential of the electrospinning technique for the encapsulation and immobilization of bacteria in the form of a synthetic biofilm, while retaining their metabolic activity. This study has wide-ranging implications in the engineering and use of novel bio-hybrid materials or biological thin-film catalysts.

Potentiometric Biosensors Based on Molecular-Imprinted Self-Assembled Monolayer Films for Rapid Detection of Influenza A Virus and SARS-CoV-2 Spike Protein.

Rapid, yet accurate and sensitive testing has been shown to be critical in the control of spreading pandemic diseases such as COVID-19. Current methods which are highly sensitive and can differentiate different strains are slow and cannot be conveniently applied at the point of care. Rapid tests, meanwhile, require a high titer and are not sufficiently sensitive to discriminate between strains. Here, we report a rapid and facile potentiometric detection method based on nanoscale, three-dimensional molecular imprints of analytes on a self-assembled monolayer (SAM), which can deliver analyte-specific detection of both whole virions and isolated proteins in microliter amounts of bodily fluids within minutes. The detection substrate with nanoscale inverse surface patterns of analytes formed by a SAM identifies a target analyte by recognizing its surface nano- and molecular structures, which can be monitored by temporal measurement of the change in substrate open-circuit potential. The sensor unambiguously detected and differentiated H1N1 and H3N2 influenza A virions as well as the spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle-East respiratory syndrome (MERS) coronavirus in human saliva with limits of detection reaching 200 PFU/mL and 100 pg/mL for the viral particles and spike proteins, respectively. The demonstrated speed and specificity of detection, combined with a low required sample volume, high sensitivity, ease of potentiometric measurement, and simple sample collection and preparation, suggest that the technique can be used as a highly effective point-of-care diagnostic platform for a fast, accurate, and specific detection of various viral pathogens and their variants.

Control of cell migration in two and three dimensions using substrate morphology.

We have shown that en masse cell migration of fibroblasts on the planar surface results in a radial outward trajectory, and a spatially dependent velocity distribution that decreases exponentially in time towards the single cell value. If the cells are plated on the surface of aligned electrospun fibers above 1 microm in diameter, they become polarized along the fiber, expressing integrin receptors which follow closely the contours of the fibers. The velocity of the cells on the fibrous scaffold is lower than that on the planar surface, and does not depend on the degree of orientation. Cells on fiber smaller than 1 microm migrate more slowly than on the planar surface, since they appear to have a large concentration of receptors. True three-dimensional migration can be observed when plating the droplet on a scaffold comprises of at least three layers. The cells still continue to migrate on the fibers surfaces, as they diffuse into the lower layers of the fibrous scaffold.

Fabrication of ordered bi-metallic array with superstructure of gold micro-rings via templated-self-assembly procedure and its SERS application.

Bi-metallic patterns with array of Pt discs decorated by Au rings were fabricated onto the substrate by a templated-self-assembly procedure in which multi-step self-assembly processes were involved. The original pattern was established by using the breath figure method. The bi-metallic sample with Au rings exhibited rather high sensitivity as well as great reproducibility within the array in surface-enhanced Raman scattering test.

Traction stresses and translational distortion of the nucleus during fibroblast migration on a physiologically relevant ECM mimic.

Cellular traction forces, resulting in cell-substrate physical interactions, are generated by actin-myosin complexes and transmitted to the extracellular matrix through focal adhesions. These processes are highly dynamic under physiological conditions and modulate cell migration. To better understand the precise dynamics of cell migration, we measured the spatiotemporal redistribution of cellular traction stresses (force per area) during fibroblast migration at a submicron level and correlated it with nuclear translocation, an indicator of cell migration, on a physiologically relevant extracellular matrix mimic. We found that nuclear translocation occurred in pulses whose magnitude was larger on the low ligand density surfaces than on the high ligand density surfaces. Large nuclear translocations only occurred on low ligand density surfaces when the rear traction stresses completely relocated to a posterior nuclear location, whereas such relocation took much longer time on high ligand density surfaces, probably due to the greater magnitude of traction stresses. Nuclear distortion was also observed as the traction stresses redistributed. Our results suggest that the reinforcement of the traction stresses around the nucleus as well as the relaxation of nuclear deformation are critical steps during fibroblast migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. A traction gradient foreshortening model was proposed to explain how the relocation of rear traction stresses leads to pulsed fibroblast migration.

Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells.

The effects of exposure of human dermal fibroblasts to rutile and anatase TiO(2) nanoparticles are reported. These particles can impair cell function, with the latter being more potent at producing damage. The exposure to nanoparticles decreases cell area, cell proliferation, mobility, and ability to contract collagen. Individual particles are shown to penetrate easily through the cell membrane in the absence of endocytosis, while some endocytosis is observed for larger particle clusters. Once inside, the particles are sequestered in vesicles, which continue to fill up with increasing incubation time till they rupture. Particles coated with a dense grafted polymer brush are also tested, and, using flow cytometry, are shown to prevent adherence to the cell membrane and hence penetration of the cell, which effectively decreases reactive oxygen species (ROS) formation and protects cells, even in the absence of light exposure. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating can potentially play an important role in protecting cells and tissue from damage.

Imaging and estimating the surface heterogeneity on a droplet containing cosolvents.

Cosolvents have numerous applications in many industries as well as scientific research. The shortage in the knowledge of the structures in a cosolvent system is significant. In this work, we display the spatial as well as the kinetic distribution of the cosolvents using droplets as paradigms. When an alcohol/water-containing sessile droplet evaporates on a substrate, it phase segregates into a water-enriched core and a thin alcohol prevailing shell. This is considered to be due to the different escaping rate of solvents out of the liquid-vapor (l-v) interfaces. In between the core and shell phases, there exists a rough and solid-like liquid-liquid (l-l) wall interface as marked by the fluorescent polystyrene spheres and imaged by a confocal microscope. Holes and patches of beads are observed to form on this phase boundary. The water-dispersed beads prefer to partition within the core. The shell prevails in the droplet during most of the drying and shrinks with the l-v boundary. By monitoring the morphological progression of the droplet, the composition of the cosolvent at the liquid-vapor interface is obtained.

In Situ Time-Resolved X-ray Scattering Study of Isotactic Polypropylene in Additive Manufacturing.

Using a specially designed apparatus, which collects simultaneous temperature and X-ray scattering data, we performed in situ measurements of the filament during MatEx 3D printing. The data show that the MatEx 3D printing extrusion process provides sufficient shear to form shish-kebab structures, which initially nucleate at the filament surface and spread into the filament core. Time-resolved measurements show that the kebab component near the surface relaxes after deposition of the second filament and enhances chain diffusion across the interface. SEM images indicate near complete interfacial merging of the filaments, which results in excellent mechanical properties.

Determination of three characteristic regimes of weakly charged polyelectrolytes monolayers.

We have demonstrated that monolayer films of randomly charged polystyrene sulfonated acid (PSSA) can be produced by the Langmuir technique, and observed the micro-domain structures, produced by the phase separation of electrostatically charged moieties and the hydrophobic moieties. Using atomic force microscopy and Langmuir isotherm, we found three specific regimes for the polyelectrolytes with various degrees of sulfonation (4-35%); very low charged PSSA (4-5%) in the hydrophobic regime, moderately charged PSSA (6-16%) which possessed a well-balanced nature between electrostatic and the hydrophobic interactions, and strongly amphiphilic nature of PSSA (6-16%) in the ionomer regime. Finally, we could categorize PSSA 35% in the polyelectrolyte regime, due to the dominance of the electrostatic interactions over the hydrophobic interactions.

Characterization of Langmuir-Blodgett organoclay films using X-ray reflectivity and atomic force microscopy.

Monolayers of organoclay platelets were formed at the air/water interface using the Langmuir technique and were then investigated either by in situ or lifted onto Si wafers and studied ex situ, using X-ray reflectivity (XR) methods. The XR data showed that the surfactant molecules on the clay platelets formed a dense, self-assembled monolayer where the molecules were tilted at an angle of 35 degrees +/-6 degrees from the normal to the dry clay surface. The surfactant layers only covered a fraction of the clay platelet surface area, where the fractional surface coverage for the three clays studied (C6A, C15A, and C20A) was found to be 0.90, 0.86, and 0.73, respectively. These values were significantly higher than those estimated from the cation exchange capacity (CEC) values. Rather than being uniformly distributed, the surfactant was clustered in patchy regions, indicating that the surface of the clay platelets had both polar and non-polar segments. This heterogeneity confirmed the hypothesis which was previously invoked to explain the distribution of the clay platelets in melt mixed homopolymer and polymer blend nanocomposites.

Multicomponent polymer coating to block photocatalytic activity of TiO2 nanoparticles.

Chemical grafting of anti-oxidant molecules with an additional hydrophobic polymer coating directly onto TiO(2) particle surfaces, using sonochemistry, is found to eliminate photocatalytic degradation enabling highly effective screening against UV radiation.

The influence of surface chemistry on adsorbed fibrinogen conformation, orientation, fiber formation and platelet adhesion.

Thrombosis is a clear risk when any foreign material is in contact with the bloodstream. Here we propose an immunohistological stain-based model for non-enzymatic clot formation that enables a facile screen for the thrombogenicity of blood-contacting materials. We exposed polymers with different surface chemistries to protease-free human fibrinogen. We observed that on hydrophilic surfaces, fibrinogen is adsorbed via αC regions, while the γ400-411 platelet-binding dodecapeptide on the D region becomes exposed, and fibrinogen fibers do not form. In contrast, fibrinogen is adsorbed on hydrophobic surfaces via the relatively hydrophobic D and E regions, exposing the αC regions while rendering the γ400-411 inaccessible. Fibrinogen adsorbed on hydrophobic surfaces is thus able to recruit other fibrinogen molecules through αC regions and polymerize into large fibrinogen fibers, similar to those formed in vivo in the presence of thrombin. Moreover, the γ400-411 is available only on the large fibers not elsewhere throughout the hydrophobic surface after fibrinogen fiber formation. When these surfaces were exposed to gel-sieved platelets or platelet rich plasma, a uniform monolayer of platelets, which appeared to be activated, was observed on the hydrophilic surfaces. In contrast, large agglomerates of platelets were clustered on fibers on the hydrophobic surfaces, resembling small nucleating thrombi. Endothelial cells were also able to adhere to the monomeric coating of fibrinogen on hydrophobic surfaces. These observations reveal that the extent and type of fibrinogen adsorption, as well as the propensity of adsorbed fibrinogen to bind platelets, may be modulated by careful selection of surface chemistry. STATEMENTS OF SIGNIFICANCE: Thrombosis is a well-known side effect of the introduction of foreign materials into the bloodstream, as might exist in medical devices including but not limited to stents, valves, and intravascular catheters. Despite many reported studies, the body's response to foreign materials in contact with the blood remains poorly understood. Current preventive methods consist of drug eluting coatings on the devices or the systemic administration of standard anticoagulants. Here we present a potential mechanism by which surface chemistry can affects fibrinogen conformation and thus affects platelet adhesion and consequently thrombus formation. Our findings suggest a possible coating which enables endothelial cell adhesion while preventing platelet adhesion.

A new strategy to engineer polymer bulk heterojunction solar cells with thick active layers via self-assembly of the tertiary columnar phase.

We report that the addition of a non-photoactive tertiary polymer phase in the binary bulk heterojunction (BHJ) polymer solar cell leads to a self-assembled columnar nanostructure, enhancing the charge mobilities and photovoltaic efficiency with surprisingly increased optimal active blend thicknesses over 300 nm, 3-4 times larger than that of the binary counterpart. Using the prototypical poly(3-hexylthiophene) (P3HT):fullerene blend as a model BHJ system, we discover that the inert poly(methyl methacrylate) (PMMA) added in the binary BHJ blend self-assembles into vertical columns, which not only template the phase segregation of electron acceptor fullerenes but also induce the out-of-plane rotation of the edge-on-orientated crystalline P3HT phase. Using complementary interrogation methods including neutron reflectivity, X-ray scattering, atomic force microscopy, transmission electron microscopy, and molecular dynamics simulations, we show that the enhanced charge transport originates from the more randomized molecular stacking of the P3HT phase and the spontaneous segregation of fullerenes at the P3HT/PMMA interface, driven by the high surface tension between the two polymeric components. The results demonstrate a potential method for increasing the thicknesses of high-performance polymer BHJ solar cells with improved photovoltaic efficiency, alleviating the burden of stringently controlling the ultrathin blend thickness during the roll-to-roll-type large-area manufacturing environment.

Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds.

A three-dimensional (3D) hyaluronic acid (HA) nanofibrous scaffold was successfully fabricated to mimic the architecture of natural extracellular matrix (ECM) based on electrospinning. Thiolated HA derivative, 3,3'-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), was synthesized and electrospun to form 3D nanofibrous scaffolds. In order to facilitate the fiber formation during electrospinning, Poly (ethylene oxide) (PEO) was added into the aqueous solution of HA-DTPH at an optimal weight ratio of 1:1. The electrospun HA-DTPH/PEO blend scaffold was subsequently cross-linked through poly (ethylene glycol)-diacrylate (PEGDA) mediated conjugate addition. PEO was then extracted in DI water to obtain an electrospun HA-DTPH nanofibrous scaffold. NIH 3T3 fibroblasts were seeded on fibronectin-adsorbed HA-DTPH nanofibrous scaffolds for 24h in vitro. Fluorescence microscopy and laser scanning confocal microscopy revealed that the 3T3 fibroblasts attached to the scaffold and spread, demonstrating an extended dendritic morphology within the scaffold, which suggests potential applications of HA-DTPH nanofibrous scaffolds in cell encapsulation and tissue regeneration.