Studying the photothermal activation of polydopamine-shelled, phase-change emulsion droplets into microbubbles using small- and ultra-small-angle neutron scattering.

Polydopamine-shelled perfluorocarbon (PDA/PFC) emulsion droplets are promising candidates for medical imaging and drug delivery applications. This study investigates their phase transition into microbubbles under near-infrared (NIR) illumination in situ using small- and ultra-small-angle neutron scattering (SANS and USANS) and contrast variation techniques. Supported by optical microscopy, thermogravimetric analysis, and ultrasound imaging, SANS and USANS results reveal rapid phase transition rates upon NIR illumination, dependent on PFC content and droplet size distribution. Specifically, perfluoropentane droplets rapidly transform into bubbles upon NIR irradiation, whereas perfluorohexane droplets exhibit greater resistance to phase change (bulk boiling points = 30 °C and 60 °C, respectively). Furthermore, smaller emulsion droplets with unimodal distribution resist NIR-triggered phase changes better than their bimodal counterparts. This observation is attributable to the lower boiling points of large emulsion droplets (lower Laplace pressure than smaller droplets) and the faster photothermal heating rates due to their thicker polydopamine shells. The insights gained from these techniques are crucial for designing phase-change emulsions activated by NIR for photothermal therapies and controlled drug delivery.

Basal spot junctions of Drosophila epithelial tissues respond to morphogenetic forces and regulate Hippo signaling.

Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. Here, we discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth.

Removal of the endothelial surface layer via hyaluronidase does not modulate monocyte and neutrophil interactions with the glomerular endothelium.

OBJECTIVE: The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. METHODS: We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. RESULTS: Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. CONCLUSIONS: These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.

Enhanced photoacoustic imaging in tissue-mimicking phantoms using polydopamine-shelled perfluorocarbon emulsion droplets.

The current work features process parameters for the ultrasound (25 kHz)-assisted fabrication of polydopamine-shelled perfluorocarbon (PDA/PFC) emulsion droplets with bimodal (modes at 100-600 nm and 1-6 µm) and unimodal (200-600 nm) size distributions. Initial screening of these materials revealed that only PDA/PFC emulsion droplets with bimodal distributions showed photoacoustic signal enhancement due to large size of their optically absorbing PDA shells. Performance of this particular type of emulsion droplets as photoacoustic agents were evaluated in Intralipid®-India ink media, mimicking the optical scattering and absorbanceof various tissuetypes. From these measurements, it was observed that PDA/PFC droplets with bimodal size distributions can enhance the photoacoustic signal of blood-mimicking phantom by up to five folds in various tissue-mimicking phantoms with absorption coefficients from 0.1 to 1.0 cm-1. Furthermore, using the information from enhanced photoacoustic images at 750 nm, the ultimate imaging depth was explored for polydopamine-shelled, perfluorohexane (PDA/PFH) emulsion droplets by photon trajectory simulations in 3D using a Monte Carlo approach. Based on these simulations, maximal tissue imaging depths for PDA/PFH emulsion droplets range from 10 to 40 mm, depending on the tissue type. These results demonstrate for the first time that ultrasonically fabricated PDA/PFC emulsion droplets have great potential as photoacoustic imaging agents that can be complemented with other reported characteristics of PDA/PFC emulsion droplets for extended applications in theranostics and other imaging modalities.

Tracking the heat-triggered phase change of polydopamine-shelled, perfluorocarbon emulsion droplets into microbubbles using neutron scattering.

Perfluorocarbon emulsion droplets are hybrid colloidal materials with vast applications, ranging from imaging to drug delivery, due to their controllable phase transition into microbubbles via heat application or acoustic droplet vapourisation. The current work highlights the application of small- and ultra-small-angle neutron scattering (SANS and USANS), in combination with contrast variation techniques, in observing the in situ phase transition of polydopamine-shelled, perfluorocarbon (PDA/PFC) emulsion droplets with controlled polydispersity into microbubbles upon heating. We correlate these measurements with optical and transmission electron microscopy imaging, dynamic light scattering, and thermogravimetric analysis to characterise these emulsions, and observe their phase transition into microbubbles. Results show that the phase transition of PDA/PFC droplets with perfluorohexane (PFH), perfluoropentane (PFP), and PFH-PFP mixtures occur at temperatures that are around 30-40 °C higher than the boiling points of pure liquid PFCs, and this is influenced by the specific PFC compositions (perfluorohexane, perfluoropentane, and mixtures of these PFCs). Analysis and model fitting of neutron scattering data allowed us to monitor droplet size distributions at different temperatures, giving valuable insights into the transformation of these polydisperse, emulsion droplet systems.

Tandem RNA binding sites induce self-association of the stress granule marker protein TIA-1.

TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3' UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.

Rapid Approach for Detection of Antibiotic Resistance in Bacteria Using Vibrational Spectroscopy.

Here, we applied vibrational spectroscopy to investigate the drug response following incubation of S. aureus with oxacillin. The main focus of this work was to identify the chemical changes caused by oxacillin over time and to determine the feasibility of the spectroscopic approach to detect antimicrobial resistance. The oxacillin-induced changes in the chemical composition of susceptible bacteria, preceding (and leading to) the inhibition of growth, included an increase in the relative content of nucleic acids, alteration in the α-helical/ß-sheet protein ratio, structural changes in carbohydrates (observed via changes in the band at 1035 cm-1), and significant thickening of the cell wall. These observations enabled a dose-dependent discrimination between susceptible bacteria incubated with and without oxacillin after 120 min. In methicillin resistant strains, no spectral differences were observed between cells, regardless of drug exposure. These results pave the way for a new, rapid spectroscopic approach to detect drug resistance in pathogens, based on their early positive/negative drug response.

Nitrogen deprivation of microalgae: effect on cell size, cell wall thickness, cell strength, and resistance to mechanical disruption.

Nitrogen deprivation (N-deprivation) is a proven strategy for inducing triacylglyceride accumulation in microalgae. However, its effect on the physical properties of cells and subsequently on product recovery processes is relatively unknown. In this study, the effect of N-deprivation on the cell size, cell wall thickness, and mechanical strength of three microalgae was investigated. As determined by analysis of micrographs from transmission electron microscopy, the average cell size and cell wall thickness for N-deprived Nannochloropsis sp. and Chlorococcum sp. were ca. 25% greater than the N-replete cells, and 20 and 70% greater, respectively, for N-deprived Chlorella sp. The average Young's modulus of N-deprived Chlorococcum sp. cells was estimated using atomic force microscopy to be 775 kPa; 30% greater than the N-replete population. Although statistically significant, these microstructural changes did not appear to affect the overall susceptibility of cells to mechanical rupture by high pressure homogenisation. This is important as it suggests that subjecting these microalgae to nitrogen starvation to accumulate lipids does not adversely affect the recovery of intracellular lipids.

p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival.

p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.

Spring constant calibration of atomic force microscope cantilevers of arbitrary shape.

The spring constant of an atomic force microscope cantilever is often needed for quantitative measurements. The calibration method of Sader et al. [Rev. Sci. Instrum. 70, 3967 (1999)] for a rectangular cantilever requires measurement of the resonant frequency and quality factor in fluid (typically air), and knowledge of its plan view dimensions. This intrinsically uses the hydrodynamic function for a cantilever of rectangular plan view geometry. Here, we present hydrodynamic functions for a series of irregular and non-rectangular atomic force microscope cantilevers that are commonly used in practice. Cantilever geometries of arrow shape, small aspect ratio rectangular, quasi-rectangular, irregular rectangular, non-ideal trapezoidal cross sections, and V-shape are all studied. This enables the spring constants of all these cantilevers to be accurately and routinely determined through measurement of their resonant frequency and quality factor in fluid (such as air). An approximate formulation of the hydrodynamic function for microcantilevers of arbitrary geometry is also proposed. Implementation of the method and its performance in the presence of uncertainties and non-idealities is discussed, together with conversion factors for the static and dynamic spring constants of these cantilevers. These results are expected to be of particular value to the design and application of micro- and nanomechanical systems in general.

MICROMORPHOGENESIS DURING DIATOM WALL FORMATION PRODUCES SILICEOUS NANOSTRUCTURES WITH DIFFERENT PROPERTIES(1).

Micromorphogenesis within the silica deposition vesicle (SDV) of the diatom Pinnularia viridis (Nitzsh) Ehrenb. resulted in distinct silica nanostructures and layers within forming valves and girdle bands. These siliceous components were similarly disclosed following alkaline etching of mature valves/girdle bands, where their different susceptibilities to dissolution over time resulted from apparent differences in silica density and/or chemistry. The bulk of silica appeared to be deposited at the interface of the forming valve or girdle band with the silicalemma and occurred by the outward expansion of microfibrils of silica that aligned perpendicularly to the silicalemma. Microfibrils originated from both sides of the "silica lamella," the first nanostructure formed within the SDV, and several silica species of distinct nanostructure and density resulted, including distinctive inner and outermost silica "coverings" of mature valves/girdle bands and the central and terminal nodules. Not all silica deposition and micromorphogenesis occurred in contact with the expanding silicalemma, but was somehow directed within the SDV cavity, and resulted in the distinct silica layers that lined the raphe fissures and poroids. Following alkaline etching, the inner surfaces of valves/girdle bands, as well as the silica layers lining the raphes, poroids, and slits, were determined to be significantly more resistant to alkaline etching than the exterior surfaces, while the outer silica coating and the nodules were quickly dissolved. The processes of micromorphogenesis must have exerted precise control over the chemical nature of the silica formed at different positions within the SDV and affected the overall structure and function of the diatom wall.

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

Characterization of the adhesive mucilages secreted by live diatom cells using atomic force microscopy.

Atomic Force Microscopy (AFM) resolved the topography and mechanical properties of two distinct adhesive mucilages secreted by the marine, fouling diatom Craspedostauros australis. Tapping mode images of live cells revealed a soft and cohesive outer mucilage layer that encased most of the diatom's siliceous wall, and force curves revealed an adhesive force of 3.58 nN. High loading force, contact mode imaging resulted in cantilever 'cleaned' cell walls, which enabled the first direct observation of the active secretion of soft mucilage via pore openings. A second adhesive mucilage consisted of strands secreted at the raphe, a distinct slit in the silica wall involved in cell-substratum attachment and motility. Force measurements revealed a raphe adhesive strand(s) resistant to breaking forces up to 60 nN, and these strands could only be detached from the AFM cantilever probe using the manual stepper motor.