Integrating Pore Architectures to Evaluate Vascularization Efficacy in Silicate-based Bioceramic Scaffolds

Pore architecture in bioceramic scaffolds plays an important role in facilitating vascularization efficiency during bone repair or orbital reconstruction. Many investigations have explored this relationship but lack integrating pore architectural features in a scaffold, hindering optimization of architectural parameters (geometry, size, curvature) to improve vascularization and consequently clinical outcomes. To address this challenge, we have developed an integrating design strategy to fabricate different pore architectures (cube, gyroid, hexagon) with different pore dimensions (∼350, 500, 650 μm) in the silicate-based bioceramic scaffolds via digital light processing technique. The sintered scaffolds maintained high-fidelity pore architectures similar to the printing model. The hexagon- and gyroid-pore scaffolds exhibited the highest and lowest compressive strength (from 15 to 55 MPa), respectively, but the cube-pore scaffolds showed appreciable elastic modulus. Moreover, the gyroid pore architecture contributed on a faster ion dissolution and mass decay in vitro. It is interesting that both μCT and histological analyses indicate vascularization efficiency was challenged even in the 650-μm pore region of hexagon-pore scaffolds within 2 weeks in rabbit models, but the gyroid pore constructs indicated appreciable blood vessel networks even in the 350-μm pore region at 2 weeks and high-density blood vessels were uniformly invaded in the 500- and 650-μm pore at 4 weeks. Angiogenesis was facilitated in the cube-pore scaffolds in comparison with the hexagon-pore ones within 4 weeks. These studies demonstrate that the continuous pore wall curvature feature in gyroid pore architecture is an important implication for biodegradation, vascular cell migration and vessel ingrowth in porous bioceramic scaffolds.

Pathogenic in-Frame Variants in SCN8A: Expanding the Genetic Landscape of SCN8A-Associated Disease

Numerous SCN8A mutations have been identified, of which, the majority are de novo missense variants. Most mutations result in epileptic encephalopathy; however, some are associated with less severe phenotypes. Mouse models generated by knock-in of human missense SCN8A mutations exhibit seizures and a range of behavioral abnormalities. To date, there are only a few Scn8a mouse models with in-frame deletions or insertions, and notably, none of these mouse lines exhibit increased seizure susceptibility. In the current study, we report the generation and characterization of two Scn8a mouse models (ΔIRL/+ and ΔVIR/+) carrying overlapping in-frame deletions within the voltage sensor of domain 4 (DIVS4). Both mouse lines show increased seizure susceptibility and infrequent spontaneous seizures. We also describe two unrelated patients with the same in-frame SCN8A deletion in the DIV S5-S6 pore region, highlighting the clinical relevance of this class of mutations.

Novel KCNQ4 variants in different functional domains confer genotype- and mechanism-based therapeutics in patients with nonsyndromic hearing loss

Loss-of-function variant in the gene encoding the KCNQ4 potassium channel causes autosomal dominant nonsyndromic hearing loss (DFNA2), and no effective pharmacotherapeutics have been developed to reverse channel activity impairment. Phosphatidylinositol 4,5-bisphosphate (PIP2), an obligatory phospholipid for maintaining KCNQ channel activity, confers differential pharmacological sensitivity of channels to KCNQ openers. Through whole-exome sequencing of DFNA2 families, we identified three novel KCNQ4 variants related to diverse auditory phenotypes in the proximal C-terminus (p.Arg331Gln), the C-terminus of the S6 segment (p.Gly319Asp), and the pore region (p.Ala271_Asp272del). Potassium currents in HEK293T cells expressing each KCNQ4 variant were recorded by patch-clamp, and functional recovery by PIP2 expression or KCNQ openers was examined. In the homomeric expression setting, the three novel KCNQ4 mutant proteins lost conductance and were unresponsive to KCNQ openers or PIP2 expression. Loss of p.Arg331Gln conductance was slightly restored by a tandem concatemer channel (WT-p.R331Q), and increased PIP2 expression further increased the concatemer current to the level of the WT channel. Strikingly, an impaired homomeric p.Gly319Asp channel exhibited hyperactivity when a concatemer (WT-p.G319D), with a negative shift in the voltage dependence of activation. Correspondingly, a KCNQ inhibitor and chelation of PIP2 effectively downregulated the hyperactive WT-p.G319D concatemer channel. Conversely, the pore-region variant (p.Ala271_Asp272del) was nonrescuable under any condition. Collectively, these novel KCNQ4 variants may constitute therapeutic targets that can be manipulated by the PIP2 level and KCNQ-regulating drugs under the physiological context of heterozygous expression. Our research contributes to the establishment of a genotype/mechanism-based therapeutic portfolio for DFNA2.

CatSper mediates the chemotactic behavior and motility of the ascidian sperm

Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration. The cation channel of sperm (CatSper), plays an important role in the regulation of intracellular Ca2+ concentration. In mammals, CatSper is the only Ca2+channel that functions in the sperm, and the mice that lack the genes for the subunits of CatSper, which make up the pore region of the Ca2+ channel, are infertile due to the inhibition of hyperactivation of the sperm. CatSper is also thought to be involved in chemotaxis in sea urchins. In contrast, in the ascidian, Ciona intestinalis, the sperm-activating and -attracting factor (SAAF) interacts with Ca2+/ATPase, which is a Ca2+-pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is the specific Ca2+channel that functions in the ascidian sperm. Therefore, in this study, we generated Catsper3 knockout (KO) animals that found that they were significantly less motile, with few motile sperms not exhibiting any chemotactic behavior. These results suggest that CatSper plays important roles in the spermatogenesis and basic motility mechanisms of sperms in both ascidians and mammals.

Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

Mapping Co-regulatory Interactions among Ligand Binding sites in RyR1

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca2+) release channel required for skeletal muscle contraction. Although cryo-electron microscopy identified binding sites of three coactivators Ca2+, ATP and caffeine (CFF), the mechanism of co-regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand binding sites and pore region in (i) Ca2+ bound-closed, (ii) ATP/CFF bound- closed, (iii) Ca2+/ATP/CFF bound-closed, and (iv) Ca2+/ATP/CFF bound-open RyR1 states. We identified two dominant interactions that mediate interactions between the Ca2+ binding site and pore region in Ca2+ bound-closed state, which partially overlapped with the pore communications in ATP/CFF bound-closed RyR1 state. In Ca2+/ATP/CFF bound-closed and -open RyR1 states, co-regulatory interactions were analogous to communications in the Ca2+ bound-closed and ATP/CFF bound- closed states. Both ATP- and CFF- binding sites mediate communication between the Ca2+ binding site and the pore region in Ca2+/ATP/CFF bound - open RyR1 structure. We conclude that Ca2+, ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation.

Modelling coating cohesion effect on soil mechanical stability and permeability of the biopore - matrix interface pore region

<p>Biopore surface is often characterized by finer particles and increased concentration of polysaccharides from root and earthworm exudates, presenting physico-chemical properties different from those of the soil matrix. Such exudates controls not only the wettability or sorption properties but also the adhesive forces of the surrounding soil particles. Thus, increased mechanical stability may be expected on biopore-matrix interface affecting preferential flow and transport processes, as well.</p> <p>However, it is still unknown (i) to what extent the particle cohesion in the coated region is able to increase the resilience of the biopore to an external loading and (ii) how it affects the permeability of the biopore-matrix pore region.</p> <p>We created a discrete element model (DEM) model of a hollow cylindrical soil sample with a coated biopore in the center (i.e., 1 cm height, 1 cm outer and 0.6 cm inner diameter). The spherical particles in the model presented diameter of 0.13 mm for the coated material and 0.22 mm for the soil matrix. The cohesion among particles in the soil matrix was set to a constant value of 10.9 MPa while the cohesion among particles in the coated region varied between 10.9 and 50.9 MPa. The sample was subjected to axial compression and the force and cracks recorded. The permeability in the radial direction from the biopore to soil matrix was calculated using ImageJ and a 3D stokes solver (FDMMS).</p> <p>The increment in the coating cohesion increased the overall soil stiffness in terms of the Young’s modulus. Before axial compression, the calculated hydraulic permeability for the interface coating and matrix was 182 μm<sup>2</sup>. After compression, although the lower coating cohesion resulted in a larger number of cracks, permeability increased with coating cohesion. This suggests that with increasing soil stiffness, the cracks decrease in number but increase in length (i.e. improved connectivity).</p>

Human ferroportin mediates proton-coupled active transport of iron

As the sole iron exporter in humans, ferroportin controls systemic iron homeostasis through exporting iron into the blood plasma. The molecular mechanism of how ferroportin exports iron under various physiological settings remains unclear. Here we found that purified ferroportin incorporated into liposomes preferentially transports Fe2+ and exhibits lower affinities of transporting other divalent metal ions. The iron transport by ferroportin is facilitated by downhill proton gradients at the same direction. Human ferroportin is also capable of transporting protons, and this activity is tightly coupled to the iron transport. Remarkably, ferroportin can conduct active transport uphill against the iron gradient, with favorable charge potential providing the driving force. Targeted mutagenesis suggests that the iron translocation site is located at the pore region of human ferroportin. Together, our studies enhance the mechanistic understanding by which human ferroportin transports iron and suggest that a combination of electrochemical gradients regulates iron export.

A Dual-Permeability Approach to Study Anomalous Moisture Transport Properties of Cement-Based Materials

Anomalous moisture transport in cement-based materials is often reported in the literature, but the conventional single-porosity moisture transport models generally fail to provide accurate simulation results. Previous studies suggested that the anomalous moisture transport could be caused by different moisture transport velocity in large and small pores. Based on this concept, the present study proposes a continuous dual-permeability model for cement-based material. The proposed model includes the transport contribution of both liquid water and water vapor, which are governed by liquid advection and vapor diffusion, respectively. We explicitly consider that moisture transport in the large pore region is faster than the small pore region. The volumetric fraction of each region is determined when fitting the measured sorption isotherms by using a bimodal equation. The validation with experimental data shows that the dual-permeability model can well simulate both the “normal” and the anomalous moisture transport. The applicability of the proposed model implies that the “dual-porosity property” could be one of reasons that cause anomalous moisture transport in cementitious materials. In addition, results show that vapor diffusion can be neglected for moisture transport in both porosities at high relative humidity (RH), while at low RH, vapor diffusion must be considered.