Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles

A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA:DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling NPs. The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mitures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole% , a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts

Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy

The Comet Interceptor Mission

Here we describe the novel, multi-point Comet Interceptor mission. It is dedicated to the exploration of a little-processed long-period comet, possibly entering the inner Solar System for the first time, or to encounter an interstellar object originating at another star. The objectives of the mission are to address the following questions: What are the surface composition, shape, morphology, and structure of the target object? What is the composition of the gas and dust in the coma, its connection to the nucleus, and the nature of its interaction with the solar wind? The mission was proposed to the European Space Agency in 2018, and formally adopted by the agency in June 2022, for launch in 2029 together with the Ariel mission. Comet Interceptor will take advantage of the opportunity presented by ESA’s F-Class call for fast, flexible, low-cost missions to which it was proposed. The call required a launch to a halo orbit around the Sun-Earth L2 point. The mission can take advantage of this placement to wait for the discovery of a suitable comet reachable with its minimum ΔV capability of 600 ms−1. Comet Interceptor will be unique in encountering and studying, at a nominal closest approach distance of 1000 km, a comet that represents a near-pristine sample of material from the formation of the Solar System. It will also add a capability that no previous cometary mission has had, which is to deploy two sub-probes – B1, provided by the Japanese space agency, JAXA, and B2 – that will follow different trajectories through the coma. While the main probe passes at a nominal 1000 km distance, probes B1 and B2 will follow different chords through the coma at distances of 850 km and 400 km, respectively. The result will be unique, simultaneous, spatially resolved information of the 3-dimensional properties of the target comet and its interaction with the space environment. We present the mission’s science background leading to these objectives, as well as an overview of the scientific instruments, mission design, and schedule

Reduced Ca²⁺ store contributes to the altered Ca²⁺ sparks in the shRNA-Bin1 fiber.

<p>Representative Fura-2 fluorescence traces of resting [Ca²⁺]_i and application of either (a) 30 mM Caffeine induced SR Ca²⁺ release in FDB fibers or (b) 5 µM Ionomycin- induced total [Ca²⁺]_i store in FDB fibers. Fiber was previously perfused with zero Ca²⁺ Tyrode before application of either caffeine or ionomycin to induce store [Ca²⁺] release. The black line represents shRNA-control fibers and the gray line represents shRNA-Bin1 fibers. The dotted line represents the level of zero fluorescence. Black dotted line in the x-axis indicates the zero values for these traces. (c) ShRNA-Bin1 fibers (blank square) (n = 20) displayed reduced mean SR and total [Ca²⁺]_i store when compared to shRNA-control fibers (filled square) (n = 25). Resting [Ca²⁺]_i levels were not affected by shRNA-Bin1.</p

shRNA-mediated knockdown of Bin1 in adult skeletal muscle.

<p>(a) Protein domains of Bin1. The listed nucleotide sequence in exon 8 represents the shRNA against Bin1 (b) Western blots of whole cell extracts from HEK293 co-transfected with a murine Bin1 cDNA plasmid and various shRNA constructs against Bin1. This assay allowed screening for the most effective shRNA at knocking down Bin1 expression. (c) High efficiency gene delivery at 14 days-post electroporation was achieved as shown by the high expression of RFP signals on the flexor digitorum brevis (FDB) muscle bundles that were electroporated with either shRNA-Bin1 or shRNA-control plasmid (<i>left</i>). RFP signal was used to identify individually transfected single muscle fiber <i>(middle</i>, <i>right</i>). (d) Western blot was performed on the trimmed FDB muscle bundle that was transfected with either shRNA-control or shRNA Bin1 to confirm the potency of shRNA-Bin1 in knocking down endogenous Bin1 expression in adult skeletal muscle (n = 4 mice).</p

Disrupted t-tubule structure in adult shRNA-Bin1 muscle.

<p>(a,<i>left</i>) Individually isolated FDB muscle fiber that was electroporated with either shRNA-control or shRNA-Bin1 plasmid was stained with 100 nM DiOC5 that labeled intact t-tubule membrane structure. shRNA-Bin1 fibers exhibited loss or diffused of t-tubule staining in shRNA-Bin1 muscle fibers (n = 13), that otherwise were not observed in the shRNA-control fibers (n = 13). Enlarged images showed that bifurcated t-tubule doublets characteristics were missing in the shRNA-Bin1 fibers (<i>middle</i>). Brightfield images <i>(right</i>) of cross-striated pattern in both shRNA-control and shRNA-Bin1 indicated healthy muscle fibers were chosen for DiOC5 staining. Several regions of interests (ROI) were drawn on the areas of the fibers that displayed blank DiOC5 staining (numerically labeled small ROIs) and then the total affected area in each fiber was divided by the total surface area of the fiber (large ROI drawn covering the surface area of the fiber). Black box represents the enlarged area showing the bifurcated t-tubule staining. (b)Quantification of missing/diffused staining observed in shRNA-Bin1 (blank square) when compared to shRNA-control (filled square) fibers. (c) EM analysis performed on the trimmed electroporated FDB muscle bundle showed that 25–30% of cells in the FDB muscle bundle transfected with shRNA-Bin1 displayed vacuolation/swollen t-tubule structure (c₁), whereas the remainder ∼70% exhibited normal t-tubule structure as seen in shRNA-control FDB muscle bundle (c₂). (c_3,4) Enlargement of the cells showing swollen t-tubule structure from the shRNA-Bin FDB muscle bundle. Arrows in (c₃) indicate normal t-tubules while in (c₄) the arrows indicate enlarged/vacuolated t-tubule structures in shRNA-Bin1 fibers. n = 56 for shRNA-control cells, and n = 63 for shRNA-Bin1 cells.</p

Defective Ca²⁺ spark signaling in shRNA-Bin1 muscle.

<p>Individually isolated FDB muscle fiber that expressed either shRNA-control or shRNA-Bin1 was treated with hypotonic osmotic stress to generate a Ca²⁺ sparks response. (a, <i>left</i>) Representative XY images of FLuo-4 fluorescence illustrated the sub-cellular localization of Ca²⁺ sparks. Images were pseudo-colored using an arbitrary scale with red appearing as the highest fluorescence signal (<i>inset</i>). shRNA-Bin1fibers (bottom) showed peripheral Ca²⁺ sparks localization despite reduced Ca²⁺ spark frequency when compared to shRNA-control (top). (a, <i>right</i>) Representative pseudocolored x<i>_t</i> linescan images of either shRNA-control or shRNA-Bin1 FDB fibers following osmotic stress. The frequency and amplitude of Ca²⁺ appear to be lower in shRNA-Bin1 fibers. (b) shRNA-Bin1 (n = 12 fibers) exhibited reduced mean Ca²⁺ spark frequency per minute when compared to shRNA-control (n = 12 fibers). (c) Correlation of the amplitude (dF/F₀) and full duration at half maximum (D₅₀) of individual Ca²⁺ release events showed a population of high amplitude and long duration Ca²⁺ release events in the shRNA-control fibers (filled square) that were absent in the shRNA-Bin1 group (blank square) (n = 973 events for shRNA-control; 1128 for shRNA-Bin1).</p