Spa2 remodels ADP-actin via molecular condensation under glucose starvation.

Actin nucleotide-dependent actin remodeling is essential to orchestrate signal transduction and cell adaptation. Rapid energy starvation requires accurate and timely reorganization of the actin network. Despite distinct treadmilling mechanisms of ADP- and ATP-actin filaments, their filament structures are nearly identical. How other actin-binding proteins regulate ADP-actin filament assembly is unclear. Here, we show that Spa2 which is the polarisome scaffold protein specifically remodels ADP-actin upon energy starvation in budding yeast. Spa2 triggers ADP-actin monomer nucleation rapidly through a dimeric core of Spa2 (aa 281-535). Concurrently, the intrinsically disordered region (IDR, aa 1-281) guides Spa2 undergoing phase separation and wetting on the surface of ADP-G-actin-derived F-actin and bundles the filaments. Both ADP-actin-specific nucleation and bundling activities of Spa2 are actin D-loop dependent. The IDR and nucleation core of Spa2 are evolutionarily conserved by coexistence in the fungus kingdom, suggesting a universal adaptation mechanism in the fungal kingdom in response to glucose starvation, regulating ADP-G-actin and ADP-F-actin with high nucleotide homogeneity.

Encapsulation of Human Spinal Cord Progenitor Cells in Hyaluronan-Gelatin Hydrogel for Spinal Cord Injury Treatment.

Transplanting human induced pluripotent stem cells (iPSCs)-derived spinal cord progenitor cells (SCPCs) is a promising approach to treat spinal cord injuries. However, stem cell therapies face challenges in cell survival, cell localization to the targeted site, and the control of cell differentiation. Here, we encapsulated SCPCs in thiol-modified hyaluronan-gelatin hydrogels and optimized scaffold mechanical properties and cell encapsulation density to promote cell viability and neuronal differentiation in vitro and in vivo. Different compositions of hyaluronan-gelatin hydrogels formulated by varying concentrations of poly(ethylene glycol) diacrylate were mechanically characterized by using atomic force microscopy. In vitro SCPC encapsulation study showed higher cell viability and proliferation with lower substrate Young's modulus (200 Pa vs 580 Pa) and cell density. Moreover, the soft hydrogels facilitated a higher degree of neuronal differentiation with extended filament structures in contrast to clumped cellular morphologies obtained in stiff hydrogels (p < 0.01). When transplanted in vivo, the optimized SCPC-encapsulated hydrogels resulted in higher cell survival and localization at the transplanted region as compared to cell delivery without hydrogel encapsulation at 2 weeks postimplantation within the rat spinal cord (p < 0.01). Notably, immunostaining demonstrated that the hydrogel-encapsulated SCPCs differentiated along the neuronal and oligodendroglial lineages in vivo. The lack of pluripotency and proliferation also supported the safety of the SCPC transplantation approach. Overall, the injectable hyaluronan-gelatin hydrogel shows promise in supporting the survival and neural differentiation of human SCPCs after transplantation into the spinal cord.

Differential Collective Cell Migratory Behaviors Modulated by Phospholipid Nanocarriers.

Phospholipid nanocarriers have been widely explored for theranostic and nanomedicine applications. These amphiphilic nanocarriers possess outstanding cargo encapsulation efficiency, high water dispersibility, and excellent biocompatibility, which render them promising for drug delivery and bioimaging applications. While the biological applications of phospholipid nanocarriers have been well documented, the fundamental aspects of the phospholipid-cell interactions beyond cytotoxicity have been less investigated. In particular, the effect of phospholipid nanocarriers on collective cell behaviors has not been elucidated. Herein, we evaluate the interactions of phospholipid nanocarriers possessing different functional groups and sizes with normal and cancerous immortalized breast epithelial cell sheets with varying metastatic potential. Specifically, we examine the impact of nanocarrier treatments on the collective migratory dynamics of these cell sheets. We observe that phospholipid nanocarriers induce differential collective cell migratory behaviors, where the migration speed of normal and cancerous breast epithelial cell sheets is retarded and accelerated, respectively. To a certain extent, the nanocarriers are able to alter the migration trajectory of the cancerous breast epithelial cells. Furthermore, phospholipid nanocarriers could modulate the stiffness of the nuclei, cytoplasm, and cell-cell junctions of the breast epithelial cell sheets, remodel their actin filament arrangement, and regulate the expressions of the actin-related proteins. We anticipate that this work will further shed light on nanomaterial-cell interactions and provide guidelines for rational and safer designs and applications of phospholipid nanocarriers for cancer theranostics and nanomedicine.

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

ABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions.

Agrin-Matrix Metalloproteinase-12 axis confers a mechanically competent microenvironment in skin wound healing.

An orchestrated wound healing program drives skin repair via collective epidermal cell proliferation and migration. However, the molecular determinants of the tissue microenvironment supporting wound healing remain poorly understood. Herein we discover that proteoglycan Agrin is enriched within the early wound-microenvironment and is indispensable for efficient healing. Agrin enhances the mechanoperception of keratinocytes by augmenting their stiffness, traction stress and fluidic velocity fields in retaliation to bulk substrate rigidity. Importantly, Agrin overhauls cytoskeletal architecture via enhancing actomyosin cables upon sensing geometric stress and force following an injury. Moreover, we identify Matrix Metalloproteinase-12 (MMP12) as a downstream effector of Agrin's mechanoperception. We also reveal a promising potential of a recombinant Agrin fragment as a bio-additive material that assimilates optimal mechanobiological and pro-angiogenic parameters by engaging MMP12 in accelerated wound healing. Together, we propose that Agrin-MMP12 pathway integrates a broad range of mechanical stimuli to coordinate a competent skin wound healing niche.

Nanovoid-Enhanced Thin-Film Composite Reverse Osmosis Membranes Using ZIF-67 Nanoparticles as a Sacrificial Template.

In this work, nanovoid-enhanced thin-film composite (TFC) membranes have been successfully fabricated using ZIF-67 nanoparticles as the sacrificial template. By incorporating different amounts of ZIF-67 during interfacial polymerization, the resultant TFC membranes can have different degrees of nanovoids after self-degradation of ZIF-67 in water, consequently influencing their physiochemical properties and separation performance. Nanovoid structures endow the membranes with additional passages for water molecules. Thus, all the newly developed TFC membranes exhibit better separation performance for brackish water reverse osmosis (BWRO) desalination than the pristine TFC membrane. The membrane made from 0.1 wt % ZIF-67 shows a water permeance of 2.94 LMH bar-1 and a salt rejection of 99.28% when being tested under BWRO at 20 bar. This water permeance is 53% higher than that of the pristine TFC membrane with the salt rejection well maintained.

High-throughput functional profiling of single adherent cells via hydrogel drop-screen.

Single-adherent-cell phenotyping on an extracellular matrix (ECM) is essential to determine cellular biological functions, such as morphological adaptations and biomolecule secretions, correlated to medical treatments and metastasis, yet there is no available platform for such high-throughput screening. Here, a novel hydrogel drop-screen device was developed to rapidly measure large-scale single-cell morphologies and multiple secretions on substrates for phenotype profiling. Single cells were first anchored to microfluidically fabricated gelatin particles providing mechanical stimulations similar to those from ECM in vivo. The cellular morphologies were then examined by quantifying the amount of cytoskeleton expressed on the particles. With droplet encapsulation, adherent single-cell multiplexed secretion analysis of a disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) was conducted at a throughput of ∼102 cells per second, revealing distinct functional heterogeneities associated with extracellular mechanical stimulations. The level of cell heterogeneity increased with increasing substrate stuffiness. Moreover, because of the promising screening capability, a database related to both nontumorigenic and tumorigenic breast cells (MCF10A, MCF-7, and MDA-MB-231) was constructed. The respective cell distributions and heterogeneities based on the morphologies and secreted bioindicators, such as MMP-2, MMP-3, MMP-9, and ADAM-8, were measured and found to correspond to the progress of tumor metastasis.

Biomimicking Fiber Platform with Tunable Stiffness to Study Mechanotransduction Reveals Stiffness Enhances Oligodendrocyte Differentiation but Impedes Myelination through YAP-Dependent Regulation.

A key hallmark of many diseases, especially those in the central nervous system (CNS), is the change in tissue stiffness due to inflammation and scarring. However, how such changes in microenvironment affect the regenerative process remains poorly understood. Here, a biomimicking fiber platform that provides independent variation of fiber structural and intrinsic stiffness is reported. To demonstrate the functionality of these constructs as a mechanotransduction study platform, these substrates are utilized as artificial axons and the effects of axon structural versus intrinsic stiffness on CNS myelination are independently analyzed. While studies have shown that substrate stiffness affects oligodendrocyte differentiation, the effects of mechanical stiffness on the final functional state of oligodendrocyte (i.e., myelination) has not been shown prior to this. Here, it is demonstrated that a stiff mechanical microenvironment impedes oligodendrocyte myelination, independently and distinctively from oligodendrocyte differentiation. Yes-associated protein is identified to be involved in influencing oligodendrocyte myelination through mechanotransduction. The opposing effects on oligodendrocyte differentiation and myelination provide important implications for current work screening for promyelinating drugs, since these efforts have focused mainly on promoting oligodendrocyte differentiation. Thus, the platform may have considerable utility as part of a drug discovery program in identifying molecules that promote both differentiation and myelination.

Thermal-Disrupting Interface Mitigates Intercellular Cohesion Loss for Accurate Topical Antibacterial Therapy.

Bacterial infections remain a leading threat to global health because of the misuse of antibiotics and the rise in drug-resistant pathogens. Although several strategies such as photothermal therapy and magneto-thermal therapy can suppress bacterial infections, excessive heat often damages host cells and lengthens the healing time. Here, a localized thermal managing strategy, thermal-disrupting interface induced mitigation (TRIM), is reported, to minimize intercellular cohesion loss for accurate antibacterial therapy. The TRIM dressing film is composed of alternative microscale arrangement of heat-responsive hydrogel regions and mechanical support regions, which enables the surface microtopography to have a significant effect on disrupting bacterial colonization upon infrared irradiation. The regulation of the interfacial contact to the attached skin confines the produced heat and minimizes the risk of skin damage during thermoablation. Quantitative mechanobiology studies demonstrate the TRIM dressing film with a critical dimension for surface features plays a critical role in maintaining intercellular cohesion of the epidermis during photothermal therapy. Finally, endowing wound dressing with the TRIM effect via in vivo studies in S. aureus infected mice demonstrates a promising strategy for mitigating the side effects of photothermal therapy against a wide spectrum of bacterial infections, promoting future biointerface design for antibacterial therapy.

Potassium channel dysfunction in human neuronal models of Angelman syndrome.

Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.

Young's Modulus Determination of Normal and Glaucomatous Human Iris.

Purpose: To evaluate the biomechanical properties (Young's modulus) of normal (control) and glaucomatous human iris using atomic force microscopy (AFM). Methods: Iris tissue obtained from eighteen glaucomatous subjects (equal number of eyes with primary angle closure glaucoma (PACG) and primary open angle glaucoma (POAG) and five normal subjects who underwent elective eye surgery were subjected to the estimation of Young's modulus by AFM. Force measurements were done at room temperature using Nanowizard II BioAFM. The iris samples were immersed in the liquid media (PBS with 0.1% BSA) during force measurements. Young's modulus values were calculated for each recorded curve using JPK Data Processing Software, which uses a Hertz's contact model for spherical indenters fitted to the extend curves. Results: The iris from the normal controls had the least Young's modulus (0.85 ± 0.31 kPa) while those from PACG patients had the highest Young's modulus (2.40 ± 0.82 kPa). The Young's modulus of PACG iris was significantly higher compared to that of the normal controls (P = 0.005) and POAG iris (P = 0.001). However, there was no significant difference in the Young's modulus of POAG iris (1.13 ± 0.36 kPa) compared to that of the normal controls (P = 0.511). Conclusions: Variations in biomechanical properties of iris tissue may have a significant role in the pathogenesis of angle closure glaucoma. This study suggests the existence of fundamental biomechanical differences in eyes with angle closure versus open angle glaucoma. An understanding of this basis creates a new platform to understand disease pathology better and work on therapeutic strategies that will address the same.

Probing the Physical Origin of Anisotropic Thermal Transport in Black Phosphorus Nanoribbons.

Black phosphorus (BP) has emerged as a promising candidate for next-generation electronics and optoelectronics among the 2D family materials due to its extraordinary electrical/optical/optoelectronic properties. Interestingly, BP shows strong anisotropic transport behavior because of its puckered honeycomb structure. Previous studies have demonstrated the thermal transport anisotropy of BP and theoretically attribute this to the anisotropy in both the phonon dispersion relation and the phonon relaxation time. However, the exact origin of such strong anisotropy lacks clarity and has yet to be proven experimentally. Here, the thermal transport anisotropy of BP nanoribbons is probed by an electron beam technique. Direct evidence is provided that the origin of this anisotropy is dominated by the anisotropic phonon group velocity, verified by Young's modulus measurements along different directions. It turns out that the ratio of the thermal conductivity between zigzag (ZZ) and armchair (AC) ribbons is almost same as that of the corresponding Young modulus values. The results from first-principles calculation are consistent with this experimental observation, where the anisotropic phonon group velocity between ZZ and AC is shown. These results provide fundamental insight into the anisotropic thermal transport in low-symmetry crystals.

Graphene oxide inhibits malaria parasite invasion and delays parasitic growth in vitro.

The interactions between graphene oxide (GO) and various biological entities have been actively investigated in recent years, resulting in numerous potential bioapplications of these nanomaterials. Despite this, the biological interactions between GO and disease-causing protozoan parasites have not been well elucidated and remain relatively unexplored. Here, we investigate the in vitro interactions between GO nanosheets and a particular species of malaria parasites, Plasmodium falciparum (P. falciparum). We hypothesize that GO nanosheets may exhibit antimalarial characteristic via action mechanisms of physical obstruction of P. falciparum parasites as well as nutrient depletion. To ascertain this, we characterize the physical interactions between GO nanosheets, red blood cells (RBCs), and malarial parasites as well as the adsorption of several biomolecules necessary for parasitic survival and growth on GO nanosheets. Subsequent to establishing the origin of this antimalarial behavior of GO nanosheets, their efficiency in inhibiting parasite invasion is evaluated. We observe that GO nanosheets at various tested concentrations significantly inhibit the invasion of malaria parasites into RBCs. Furthermore, GO nanosheets delay parasite progression from the ring to the trophozoite stage. Overall, this study may further shed light on the graphene-parasite interactions and potentially facilitate the development of nanomaterial-based strategies for combating malaria.

Reinforcing Low-Volume Fraction Nano-TiN Particulates to Monolithical, Pure Mg for Enhanced Tensile and Compressive Response.

Novel Mg (0.58, 0.97, 1.98 and 2.5) vol. % TiN nanocomposites containing titanium nitride (TiN) nanoparticulates of ~20 nm size are successfully synthesized by a disintegrated melt deposition technique followed by hot extrusion. Microstructural characterization of Mg-TiN nanocomposites indicate significant grain refinement with Mg 2.5 vol. % TiN exhibiting a minimum grain size of ~11 µm. X-ray diffraction studies of Mg-TiN nanocomposites indicate that addition of up to 1.98 vol. % TiN nanoparticulates aids in modifying the strong basal texture of pure Mg. An attempt is made to study the effects of the type of titanium (metal or ceramic), size, and volume fraction addition of nanoparticulates on the microstructural and mechanical properties of pure magnesium. Among the major strengthening mechanisms contributing to the strength of Mg-Ti-based nanocomposites, Hall-Petch strengthening was found to play a vital role. The synthesized Mg-TiN nanocomposites exhibited superior tensile and compression properties indicating significant improvement in the fracture strain values of pure magnesium under loading. Under tensile and compression loading the presence of titanium (metal or ductile phase) nanoparticulates were found to contribute more towards the strengthening, whereas ceramics of titanium (brittle phases) contribute more towards the ductility of pure magnesium.

Role of Cytoskeletal Tension in the Induction of Cardiomyogenic Differentiation in Micropatterned Human Mesenchymal Stem Cell.

The role of biophysical induction methods such as cell micropatterning in stem cell differentiation has been well documented previously. However, the underlying mechanistic linkage of the engineered cell shape to directed lineage commitment remains poorly understood. Here, it is reported that micropatterning plays an important role in regulating the optimal cytoskeletal tension development in human mesenchymal stem cell (hMSC) via cell mechanotransduction pathways to induce cardiomyogenic differentiation. Cells are grown on fibronectin strip patterns to control cell polarization and morphology. These patterned cells eventually show directed commitment toward the myocardial lineage. The cell's mechanical properties (cell stiffness and cell traction forces) are observed to be very different for cells that have committed to the myocardial lineage when compared with that of control. These committed cells have mechanical properties that are significantly lower indicating a correlation between the micropatterning-induced differentiation and actomyosin-generated cytoskeletal tension within patterned cells. To study this correlation, patterned cells are treated with RhoA pathway inhibitor. Severely down-regulated cardiomyogenic marker expression is observed in those treated patterned cells, thus emphasizing the direct dependence of hMSCs differentiation fate on the cytoskeletal tension.

Rapid, high-throughput tracking of bacterial motility in 3D via phase-contrast holographic video microscopy.

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility.

Effects of Primary Processing Techniques and Significance of Hall-Petch Strengthening on the Mechanical Response of Magnesium Matrix Composites Containing TiO2 Nanoparticulates.

In the present study, Mg (1.98 and 2.5) vol % TiO2 nanocomposites are primarily synthesized utilizing solid-phase blend-press-sinter powder metallurgy (PM) technique and liquid-phase disintegrated melt deposition technique (DMD) followed by hot extrusion. Microstructural characterization of the synthesized Mg-TiO2 nanocomposites indicated significant grain refinement with DMD synthesized Mg nanocomposites exhibiting as high as ~47% for 2.5 vol % TiO2 NPs addition. X-ray diffraction studies indicated that texture randomization of pure Mg depends not only on the critical amount of TiO2 NPs added to the Mg matrix but also on the adopted synthesis methodology. Irrespective of the processing technique, theoretically predicted tensile yield strength of Mg-TiO2 nanocomposites was found to be primarily governed by Hall-Petch mechanism. Among the synthesized Mg materials, solid-phase synthesized Mg 1.98 vol % TiO2 nanocomposite exhibited a maximum tensile fracture strain of ~14.5%. Further, the liquid-phase synthesized Mg-TiO2 nanocomposites exhibited higher tensile and compression properties than those primarily processed by solid-phase synthesis. The tensile-compression asymmetry values of the synthesized Mg-TiO2 nanocomposite was found to be lower than that of pure Mg with solid-phase synthesized Mg 1.98 vol % TiO2 nanocomposite exhibiting as low as 1.06.

Polysaccharide nanofibers with variable compliance for directing cell fate.

Cells perceive their microenvironment through physical and mechanical cues, such as extracellular matrix topography or stiffness. In this study, we developed a polysaccharide scaffold that can provide combined substrate topography and matrix compliance signals to direct cell fate. Pullulan/dextran (P/D) nanofibers were fabricated with variable stiffness by in situ crosslinking during electrospinning. By varying the chemical crosslinking content between 10, 12, 14, and 16%, (denoted as STMP10, STMP12, STMP14, and STMP16 respectively), scaffold mechanical stiffness was altered. We characterized substrate stiffness by various methods. Under hydrated conditions, atomic force microscopy and tensile tests of bulk scaffolds were conducted. Under dry conditions, tensile tests of scaffolds and single nanofibers were examined. In addition, we evaluated the efficacy of the scaffolds in directing stem cell differentiation. Using human first trimester mesenchymal stem cells (fMSCs) cultured on STMP14 P/D scaffolds (Young's modulus: 7.84 kPa) in serum-free neuronal differentiation medium exhibited greatest extent of differentiation. Cells showed morphological changes and significantly higher expression of motor neuron markers. Further analyses by western blotting also revealed the enhanced expression of choline acetyltransferase on STMP14 (7.84 kPa) and STMP16 (11.08 kPa) samples as compared to STMP12 (7.19 kPa). Taken together, this study demonstrates that the stiffness of P/D nanofibers can be altered by differential in situ crosslinking during electrospinning and suggests the feasibility of using such polysaccharide nanofibers in supporting fMSC neuronal commitment.

Epithelial bridges maintain tissue integrity during collective cell migration.

The ability of skin to act as a barrier is primarily determined by the efficiency of skin cells to maintain and restore its continuity and integrity. In fact, during wound healing keratinocytes migrate collectively to maintain their cohesion despite heterogeneities in the extracellular matrix. Here, we show that monolayers of human keratinocytes migrating along functionalized micropatterned surfaces comprising alternating strips of extracellular matrix (fibronectin) and non-adherent polymer form suspended multicellular bridges over the non-adherent areas. The bridges are held together by intercellular adhesion and are subjected to considerable tension, as indicated by the presence of prominent actin bundles. We also show that a model based on force propagation through an elastic material reproduces the main features of bridge maintenance and tension distribution. Our findings suggest that multicellular bridges maintain tissue integrity during wound healing when cell-substrate interactions are weak and may prove helpful in the design of artificial scaffolds for skin regeneration.