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Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.

Cluster velocity and slip velocity are the important features of cluster hydrodynamics and the statistical analysis was carried out based on the moving direct of the clusters in this work. Lagrange approach was employed to compute the velocity of the clusters which were captured by a high speed camera in a thin 2D fluidized bed. The experimental research results show that cluster velocity can be accurately computed by the Lagrange method. The velocity of the up-moving clusters is slightly greater than that of down-moving cluster. Cluster velocity increases with increasing the gas velocity and most of cluster velocity ranges from −2 to 2 m/s. The smaller clusters have greater cluster velocity and the larger cluster have smaller cluster velocity. Most of the down-moving clusters are present in the near wall region, and the down-moving clusters are about 60–70% of the total clusters. The x-component of cluster velocity is low, ranging from 0.3 to 0.6 m/s. Cluster slip velocity reveals the interaction between the cluster and its surrounding gas, and is an essential parameter of the structure-based model in the gas-solid fluidized bed. The distribution of cluster slip velocity is wider than that of cluster velocity, and they have the similar various trends. A small amount of slip velocity is negative, about 20–30%, for the up-moving clusters, while all of slip velocity is positive for down-moving clusters.

Summary Emerging evidence challenges the lens as an immune-privileged organ. Here, we provide a direct mechanism supporting a role of macrophages in lens capsule rupture repair. Posterior lens capsule rupture in a connexin 50 and aquaporin 0 double-knockout mouse model resulted in lens tissue extrusion into the vitreous cavity with formation of a “tail-like” tissue containing delayed regressed hyaloid vessels, fibrotic tissue and macrophages at postnatal (P) 15 days. The macrophages declined after P 30 days with M2 macrophages detected inside the lens. By P 90 days, the “tail-like” tissue completely disappeared and the posterior capsule rupture was sealed with thick fibrotic tissue. Colony-stimulating factor 1 (CSF-1) accelerated capsule repair, whereas inhibition of the CSF-1 receptor delayed the repair. Together, these results suggest that lens posterior rupture leads to the recruitment of macrophages delivered by the regression delayed hyaloid vessels. CSF-1-activated M2 macrophages mediate capsule rupture repair and development of fibrosis., Graphical abstract, Highlights • Lens posterior rupture delays regression of the hyaloid vessels. • Lens posterior rupture recruits macrophages delivered by the hyaloid vessels. • Macrophages mediate necrotic fiber cell removal and capsule rupture sealing. • CSF-1 activated M2 macrophages facilitate capsular rupture sealing by fibrosis., Immunology; Ophthalmology

Summary Macrophage (MΦ) activation and promotion of fibrosis are critical processes in lens capsule healing after injury. Here, we detail a protocol that induces MΦ2 formation within the vitreous body of the eye. Our procedure combines the use of an intravitreal injection of a growth factor (CSF-1) and immunofluorescence to confirm the presence of MΦ2 and fibrotic tissue formation. This protocol allows assessment of the distribution of macrophages and quantification of fibrotic tissue formation/sealing within the vitreous body of mouse eyes. For complete details on the use and execution of this profile, please refer to Li et al. (2021), Gerhardt et al. (2003), Kubota et al. (2009)., Graphical abstract, Highlights • Use of intravitreal injection of CSF-1 protein to topically activate macrophages • Immunofluorescence assessment of different MΦ subtypes simultaneously • Study of macrophage activation in the vitreous body and lens, Macrophage (MΦ) activation and promotion of fibrosis are critical processes in lens capsule healing after injury. Here, we detail a protocol that induces MΦ2 formation within the vitreous body of the eye. Our procedure combines the use of an intravitreal injection of a growth factor (CSF-1) and immunofluorescence to confirm the presence of MΦ2 and fibrotic tissue formation. This protocol allows assessment of the distribution of macrophages and quantification of fibrotic tissue formation/sealing within the vitreous body of mouse eyes.

Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2+-dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46–CaM interaction and attenuated its abolishment by Ca2+ depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2+-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2+ sensitivity. The data suggest that the G143R-induced enhancement of the CaM–Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.

To improve the accuracy of simulating the heterogeneous flow in a circulating fluidized bed (CFB) downer, a drag model based on the local structure of the gas-solid flow using the multi-scale method has been developed in this work. New stability conditions according to the characteristics of gas-solid flow in the CFB downer have been derived to solve the non-linear model equations for obtaining structure parameters. New structure-based drag coefficients were incorporated into the two-fluid model (TFM) to simulate the hydrodynamics of the gas-solid flow in the downer. Simulation results showed that the predictions with the new structure-based drag model are more accurate than those with the Wen-Yu drag model. The predictions with the structure-based drag model showed the heterogeneous flow of the gas-solid flow and the cluster phenomenon. The fluctuation of the instantaneous solid fraction of the experiment can be reasonably reproduced by the simulation using the new structure-based drag model, whereas the fluctuation of the instantaneous solid fraction cannot be captured by the Wen-Yu drag model. The experimental data showed that the radial profiles of the solid fraction are a typical core-annular structure, and the simulated results with new structure-based drag model agreed well with experimental data from different cases. The axial profiles of the simulated pressure gradient, solid concentration and particles velocity distinctly exhibit the axial structure of the gas-solid flow in the downer, which agreed with the experimental results.

Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone.

Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue ∼242 to ∼335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.

We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four alpha-helices and Val(362) was found to be located in the middle of the 3rd helix. Replacement of Val(362) with amino acid residues that disrupt the alpha-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the alpha-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val(362) is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation.

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu(367) and Gly(368). We have shown previously that Ser(363) is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser(363) was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.