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A low-calcium microenvironment is essential for spermatozoa to mature in the epididymis; however, it remains unclear how dysregulation of epididymal luminal calcium is associated with male infertility. Using a warfarin-induced vitamin K2 deficiency rat model, we found that vitamin-K-dependent γ-glutamyl carboxylase (GGCX) and matrix Gla protein (MGP) were essential in extracellular calcium signaling of the intercellular communication required for epididymal sperm maturation. We found that GGCX and MGP co-localized in the vesicular structures of epididymal cells and spermatozoa. Calcium-regulated MGP binds to proteins in a biphasic manner; sub-millimolar calcium enhances, whereas excessive calcium inhibits, the binding. Bioinformatic analysis of the calcium-dependent MGP-bound proteome revealed that vesicle-mediated transport and membrane trafficking underlie the intercellular communication networks. We also identified an SNP mutation, rs699664, in the GGCX gene of infertile men with asthenozoospermia. Overall, we revealed that the GGCX-MGP system is integrated with the intercellular calcium signaling to promote sperm maturation., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antiangiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). However, the only clinically approved systemic antiangiogenic agent for advanced HCC is sorafenib, which exerts considerable toxicity. Moreover, acquired resistance to antiangiogenic therapy often develops and restricts the therapeutic efficacy of this treatment. Hence, in this study, we develop a CXCR4-targeted lipid-based nanoparticle (NP) formulation to specifically deliver vascular endothelial growth factor (VEGF) siRNA as an antiangiogenic substance into HCC. AMD3100, a CXCR4 antagonist, is added into NPs to serve as both a targeting moiety and a sensitizer to antiangiogenic therapy. We demonstrate that AMD-modified NPs (AMD-NPs) can efficiently deliver VEGF siRNAs into HCC and downregulate VEGF expression in vitro and in vivo. Despite the upregulation of the SDF1α/CXCR4 axis upon the induction of hypoxia after antiangiogenic therapy, CXCR4 inhibition by AMD-NPs in combination with either conventional sorafenib treatment or VEGF siRNA prevents the infiltration of tumor-associated macrophages. These dual treatments also induce synergistic antiangiogenic effects and suppress local and distant tumor growth in HCC. In conclusion, the tumor-targeted multifunctional AMD-NPs that co-deliver VEGF siRNA and AMD3100 provide an effective approach for overcoming tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.