Negative charges in the flexible N-terminal domain of Rho GDP-dissociation inhibitors (RhoGDIs) regulate the targeting of the RhoGDI-Rac1 complex to membranes.

In its resting state, Rho GDP-dissociation inhibitor (RhoGDI) α forms a soluble cytoplasmic heterodimer with the GDP-bound form of Rac. Upon stimulation, the dissociation of RhoGDIα from the RhoGDIα-Rac complex is a mandatory step for Rac activation; however, this mechanism is poorly understood. In this study, we examined how the cytoplasm/membrane cycles of the RhoGDI-Rac complex are regulated, as well as where RhoGDI dissociates from the RhoGDI-Rac complex, during FcγR-mediated phagocytosis. The negatively charged and flexible N terminus (25 residues) of RhoGDIα, particularly its second negative amino acid cluster possessing five negatively charged amino acids, was a pivotal regulator in the cytoplasm/membrane cycles of the RhoGDI-Rac complex. We also found that RhoGDIα translocated to the phagosomes as a RhoGDIα-Rac1 complex, and this translocation was mediated by an interaction between the polybasic motif in the C terminus of Rac1 and anionic phospholipids produced on phagosomes, such as phosphatidic acid, that is, by a phagosome-targeting mechanism of Rac1. Thus, we demonstrated that the targeting/accumulation of the RhoGDIα-Rac1 complex to phagosomes is regulated by a balance between three factors: 1) the negatively charged and flexible N-terminal of RhoGDIα, 2) the binding affinity of RhoGDIα for Rac1, and 3) anionic phospholipids produced on phagosomes. Moreover, we demonstrated that the mechanism of targeting/accumulation of the RhoGDIα-Rac1 complex is also applicable for the RhoGDIß-Rac1 complex.

The effect of sacroiliac joint mobilization on pelvic deformation and the static balance ability of female university students with si joint dysfunction.

[Purpose] The present study aimed to determine the effect of an 8-week program of joint mobilization on changes in pelvic obliquity and pain level in seventeen female university students aged in their 20's with sacroiliac joint dysfunction by dividing them into two groups: a joint mobilization group (MWM) and a control group. [Subjects] Seventeen subjects were selected from female university students aged in their 20's attending N University in Cheon-An City, Korea, The subjects had sacroiliac joint syndrome, but experienced no problems with daily living and had no previous experience of joint mobilization exercise. The subjects were randomly assigned to a joint mobilization group of eight and a control group of nine who performed joint mobilization exercise. [Methods] Body fat and lean body mass were measured using InBody 7.0 (Biospace, Korea). The Direct Segmental Multi-frequency Bioelectrical Impedance Analysis Method (DSM-BIA) was used for body composition measurement. A pressure footstool (Pedoscan, DIERS, Germany) and a trunk measurement system (Formetric 4D, DIERS, Germany), a 3D image processing apparatus with high resolution for vertebrae, were used to measure 3D trunk images of the vertebrae and pelvis obliquity, as well as static balance ability. [Result] The MWM group showed a significantly better Balance than the control group. In addition, the results of the left/right and the front/rear balance abilities were significantly better than those of the control group. [Conclusion] This study proved that a combination of mobilization with movement and functional training was effective in reducing pelvis malposition and pain, and improving static stability control.

Bioprinting of pre-vascularized constructs for enhancedin vivoneo-vascularization.

Pre-vascularization has been receiving significant attention for developing implantable engineered 3D tissues. While various pre-vascularization techniques have been developed to improve graft vascularization, the effect of pre-vascularized patterns onin vivoneo-vessel formation has not been studied. In this study, we developed a functional pre-vascularized construct that significantly promotes graft vascularization and conductedin vivoevaluations of the micro-vascular patterns (µVPs) in various printed designs.µVP formation, composed of high-density capillaries, was induced by the co-printing of endothelial cells and adipose-derived stem cells (ADSC). We implanted the printed constructs with variousµVP designs into a murine femoral arteriovenous bundle model and evaluated graft vascularization via 3D visualization and immune-histological analysis of the neo-vessels. TheµVP-distal group (µVP located away from the host vessel) showed approximately two-fold improved neo-vascularization compared to theµVP-proximal group (µVP located near the host vessel). Additionally, we confirmed that theµVP-distal group can generate the angiogenic factor gradient spatial environment for graft vascularization via computational simulations. Based on these results, the ADSC mono pattern (AMP), which secretes four times higher angiogenic factors thanµVP, was added to theµVP + AMP group design. TheµVP + AMP group showed approximately 1.5- and 1.9-fold higher total sprouted neo-vessel volume than theµVP only and AMP only groups, respectively. In immunohistochemical staining analysis, theµVP + AMP group showed two-fold improved density and diameter of the matured neo-vessels. To summarize, these findings demonstrate graft vascularization accelerated due to design optimization of our pre-vascularized constructs. We believe that the developed pre-vascularization printing technique will facilitate new possibilities for the upscaling of implantable engineered tissues/organs.

Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding.

During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.

Application of concentrated deep sea water inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice.

BACKGROUND: Mineral water from deep-sea bedrock, formed over thousands of years, is rich in minerals such as Ca, Mg, Na, K, Fe and others. Our present study was to investigate the preventive effects of natural deep-sea water on developing atopic dermatitis (AD). METHODS: We elicited AD by application of DNCB (2,4-dinitro-chlorobezene) in Nc/Nga mouse dorsal skin. Deep Sea water (DSW) was filtered and concentrated by a nanofiltration process and reverse osmosis. We applied concentrated DSW (CDSW) to lesions five times per week for six weeks, followed by evaluation. 1% pimecrolimus ointment was used as positive control. The severity of skin lesions was assessed macroscopically and histologically. Levels of inflammatory mediators and cytokines in the serum were detected by Enzyme-linked immunosorbent assay (ELISA) and the levels of CD4+ and CD8+ spleen lymphocytes were determined by flow cytometry analysis. RESULTS: DNCB-treated mice showed atopic dermatitis-like skin lesions. Treatment of mice with CDSW reduced the severity of symptoms in the skin lesions, including edema, erythema, dryness, itching, and transepidermal water loss (TEWL). Histological analyses demonstrated that epidermal thickness and infiltration of inflammatory cells were decreased after CDSW treatment. Given these interesting observations, we further evaluated the effect of CDSW on immune responses in this AD model. Treatment AD mice with CDSW inhibited up-regulation of IgE, histamine, and pro-inflammatory cytokines in the serum. Also, the CD4+/CD8+ ratio in spleen lymphocyte was down-regulated after treatment with CDSW. Finally, cytokines, especially IL-4 and IL-10 which are important for Th2 cell development, were reduced. CONCLUSIONS: Our data suggests that topical application of CDSW could be useful in preventing the development of atopic dermatitis.

Development of Müller cell-based 3D biomimetic model using bioprinting technology.

Müller cells are the principal glial cells for the maintenance of structural stability and metabolic homeostasis in the human retina. Although variousin vitroexperiments using two-dimensional (2D) monolayer cell cultures have been performed, the results provided only limited results because of the lack of 3D structural environment and different cellular morphology. We studied a Müller cell-based 3D biomimetic model for use in experiments on thein vivo-like functions of Müller cells within the sensory retina. Isolated primary Müller cells were bioprinted and a 3D-aligned architecture was induced, which aligned Müller cell structure in retinal tissue. The stereographic and functional characteristics of the biomimetic model were investigated and compared to those of the conventional 2D cultured group. The results showed the potential to generate Müller cell-based biomimetic models with characteristic morphological features such as endfeet, soma, and microvilli. Especially, the 3D Müller cell model under hyperglycemic conditions showed similar responses as observed in thein vivodiabetic model with retinal changes, whereas the conventional 2D cultured group showed different cytokine and growth factor secretions. These results show that our study is a first step toward providing advanced tools to investigate thein vivofunction of Müller cells and to develop complete 3D models of the vertebrate retina.

Engineering Tissue-Specific, Multiscale Microvasculature with a Capillary Network for Prevascularized Tissue.

Although there are various pre-existing technologies for engineering vasculatures, multiscale modeling of the architecture of human vasculature at a capillary scale remains a challenge. In this study, a novel technology is developed for the production of a functional, multiscale microvasculature comprising of endothelialized channels and tissue-specific capillary networks. Perfusable, endothelialized channels are bioprinted, after which angiogenic sprouts are grown into user-designed capillary networks. The induction of branched and liver-lobule-like capillary networks confirm that the technology can produce various types of tissue-specific multiscale microvasculatures. Further, the channels and capillaries are deemed to be functional when evaluated in vitro. An ex vivo assay demonstrates that the microvasculature can induce neovessel ingrowth, integrate with host vessels, and facilitate blood flow. Remarkably, blood flows through the implanted capillary network without any change in its morphology. Finally, the technology is applied to produce a vascularized liver tissue; it significantly improves its hepatic function. It is believed that this new technology will create new possibilities in the development of highly vascularized and functional tissues/organs on a clinically relevant scale.

In Vitro Mechanical and Biological Properties of 3D Printed Polymer Composite and ß-Tricalcium Phosphate Scaffold on Human Dental Pulp Stem Cells.

3D printed biomaterials have been extensively investigated and developed in the field of bone regeneration related to clinical issues. However, specific applications of 3D printed biomaterials in different dental areas have seldom been reported. In this study, we aimed to and successfully fabricated 3D poly (lactic-co-glycolic acid)/ß-tricalcium phosphate (3D-PLGA/TCP) and 3D ß-tricalcium phosphate (3D-TCP) scaffolds using two relatively distinct 3D printing (3DP) technologies. Conjunctively, we compared and investigated mechanical and biological responses on human dental pulp stem cells (hDPSCs). Physicochemical properties of the scaffolds, including pore structure, chemical elements, and compression modulus, were characterized. hDPSCs were cultured on scaffolds for subsequent investigations of biocompatibility and osteoconductivity. Our findings indicate that 3D printed PLGA/TCP and ß-tricalcium phosphate (ß-TCP) scaffolds possessed a highly interconnected and porous structure. 3D-TCP scaffolds exhibited better compressive strength than 3D-PLGA/TCP scaffolds, while the 3D-PLGA/TCP scaffolds revealed a flexible mechanical performance. The introduction of 3D structure and ß-TCP components increased the adhesion and proliferation of hDPSCs and promoted osteogenic differentiation. In conclusion, 3D-PLGA/TCP and 3D-TCP scaffolds, with the incorporation of hDPSCs as a personalized restoration approach, has a prospective potential to repair minor and critical bone defects in oral and maxillofacial surgery, respectively.

Inhibitory effect of ginsenosides from steamed ginseng-leaves and flowers on the LPS-stimulated IL-12 production in bone marrow-derived dendritic cells.

Interleukin-12, a heterodimeric cytokine comprising p40 and p35 subunits, plays an essential role in the regulating the differentiation of Th cells, which establish and maximize the capabilities of the immune system. The aim of present study is to screen the effect of 21 ginsenosides from steamed ginseng-leaves and flowers on IL-12 production in bone marrow-derived dendritic cells induced by lipopolysaccharide. Noticeably, ginsenoside Rg(6) (12) and ginsenoside F(4) (13) exhibited particularly inhibitory effect on LPS-induced IL-12 production with the inhibition values of 80 and 82%; and ginsenoside ST(1) (4), ginsenoside SL(2) (8), ginsenoside SL(3) (9), ginsenoside Rh(3) (14), ginsenoside Rk(2) (15), and ginsenoside Rs(4) (18) showed moderate effects with inhibition rates of 63, 65, 67, 68, 71, 73, and 67%, respectively. These results warrant further studies concerning potential of saponin extracts of steamed ginseng-leaves and flowers for medicinal uses.

Sequential binding of cytosolic Phox complex to phagosomes through regulated adaptor proteins: evaluation using the novel monomeric Kusabira-Green System and live imaging of phagocytosis.

We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40phox interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47phox and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341-360. Furthermore, we demonstrated sequential interactions of p67phox with phagosomes involving adaptor proteins, p47phox and p40phox, during FcgammaR-mediated phagocytosis. Although p67phox is not targeted to phagosomes by itself, p47phox functions as an adaptor for the ternary complex (p47phox-p67phox-p40phox) in early stages of phagocytosis before phagosome closure, while p40phox functions in later stages after phagosomal closure. Interestingly, a mutated "open" form of p40phox linked p47phox to closed phagosomes and prolonged p47phox and p67phox retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.