Ductal delivery of extracellular vesicles promote the recovery from salivary gland inflammation.

Salivary gland dysfunction worsens the quality of life, but treatment for restoration of salivary gland function is limited. Although previous reports have demonstrated the therapeutic potentials of extracellular vesicles (EVs) in different preclinical models, the role of EVs in salivary glands remains elusive. Furthermore, little is known about the roles of salivary gland-derived EVs in tissue repair or regeneration compared to other EVs. In this study, EVs secreted from salivary gland-derived mesenchymal stem cells (sgMSCs) were comparatively analyzed with those from Wharton's jelly-derived MSC (wjMSCs). sgMSCs secreted more significant amounts of EVs than wjMSCs, and salivary gland epithelial cells showed a more efficient uptake of sgMSC-EVs than wjMSC-EVs. The possibility of immune regulation was tested via macrophage polarization and LPS-induced epithelial inflammation, resulting in an M1-to-M2 shift and reversal of acinar-to-ductal metaplasia by sgMSC-EV. Furthermore, the roles of sgMSC-EV-mediated immune regulation and tissue repair were clarified in vivo via retroductal delivery of sgMSC-EVs in a mouse model of obstructive sialadenitis. Collectively, our data demonstrate the superior role of sgMSC-EVs in the recovery from salivary gland inflammation and injury and suggest EVs as therapeutic tools for salivary gland dysfunction.

Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glands.

Salivary glands that produce and secrete saliva, which is essential for lubrication, digestion, immunity, and oral homeostasis, consist of diverse cells. The long-term maintenance of diverse salivary gland cells in organoids remains problematic. Here, we establish long-term murine and human salivary gland organoid cultures. Murine and human salivary gland organoids express gland-specific genes and proteins of acinar, myoepithelial, and duct cells, and exhibit gland functions when stimulated with neurotransmitters. Furthermore, human salivary gland organoids are established from isolated basal or luminal cells, retaining their characteristics. Single-cell RNA sequencing also indicates that human salivary gland organoids contain heterogeneous cell types and replicate glandular diversity. Our protocol also enables the generation of tumoroid cultures from benign and malignant salivary gland tumor types, in which tumor-specific gene signatures are well-conserved. In this study, we provide an experimental platform for the exploration of precision medicine in the era of tissue regeneration and anticancer treatment.

17Beta-Estradiol Regulates NUCB2/ Nesfatin-1 Expression in Mouse Oviduct.

NUCB2/nesfatin-1 known to regulate appetite and energy homeostasis is expressed not only in the hypothalamus, but also in various organs and tissues. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the reproductive organs, including the ovaries, uterus, and testes of mice. However, it is yet known whether NUCB2/nesfatin-1 is expressed in the oviduct and how its expression is regulated. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the oviduct and its expression is regulated by gonadotropin. Immunohistochemical staining results showed that nesfatin-1 protein was localized in epithelial cells of the oviduct. As a result of quantitative real-time PCR (qRT-PCR) and Western blot, NUCB2/nesfatin-1 was detected strongly in the oviducts. During the estrus cycle, NUCB2/nesfatin-1 expression in the oviducts was markedly higher in the proestrus stage than in other estrus stages. In order to elucidate whether the expression of NUCB2 mRNA is controlled by the gonadotropins, we injected PMSG and hCG and measured NUCB2 mRNA level in the oviduct after injection. Its level was increased in the oviduct after PMSG injection, but no significant change after hCG injection. In addition, NUCB2 mRNA levels were markedly reduced after ovariectomy, while recovered after 17ß-estradiol (E2) injection, but not by progesterone (P4). This study demonstrated that NUCB2/nesfatin-1 is highly expressed in the oviduct of mouse and its expression is regulated by E2 secreted by the ovaries. These results suggest that NUCB2/nesfatin-1 expressed by the oviduct may affect the function of the oviduct regulated by the ovaries.

Surface potential microscopy of surfactant-controlled single gold nanoparticle.

The surface potential of nanoparticles plays a key role in numerous applications, such as drug delivery and cellular uptake. The estimation of the surface potential of nanoparticles as drug carriers or contrast agents is important for the design of nanoparticle-based biomedical platforms. Herein, we report the direct measurement of the surface potential of individual gold nanorods (GNRs) via Kelvin probe force microscopy (KPFM) at the nanoscale. GNRs were capped by a surfactant, cetyltrimethylammonium bromide (CTAB), which was removed by centrifugation. CTAB removal is essential for GNR-based biomedical applications because of the cytotoxicity of CTAB. Applying KPFM analysis, we found that the mean surface potential of the GNRs became more negative as the CTAB was removed from the GNR. The results indicate that the negative charge of GNRs is covered by the electrostatic charge of the CTAB molecules. Similar trends were observed in experiments with gold nanospheres (GNS) capped by citrates. Overall, KPFM-based techniques characterize the surfactant of individual nanoparticles (i.e. GNR or GNS) with high resolution by mapping the surface potential of a single nanoparticle, which aids in designing engineered nanoparticles for biomedical applications.

In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model.

A magnetic polyaniline nanohybrid for MR imaging and redox sensing of cancer cells.

A synthetic process for constructing an organo-metal nanohybrid is described. This process uses polyaniline as a ligand in order to fabricate magnetic nanoparticles. This nanohybrid shows imaging potential uses as a magnetic resonance imaging contrast agent and a redox-sensing probe simultaneously both in vitro and in vivo.

Maleimidyl magnetic nanoplatform for facile molecular MRI.

In this study, we developed the maleimidyl magnetic nanoplatform, which enables functional targeting of a biomarker-specific moiety for molecular imaging via MRI. The maleimide group of the maleimidyl magnetic nanoplatform is conjugated with a thiol group without additional crosslinkers and side products. A physicochemical analysis was conducted to verify the effectiveness of the maleimidyl magnetic nanoplatform, and the existence of the maleimidyl group was investigated using the platform. To prepare biomarker-specific MRI probes, a thiolated aptamer and peptide were immobilized onto the maleimidyl group of the maleimidyl magnetic nanoplatform. The fabricated MRI probes were applied to four cancer cell lines: HT1080, MCF7, MKN45, and HEK293T. To investigate the potential of the molecular MRI probe, the target-biomarker specificity was confirmed without serious cytotoxicity, and in vivo MRI analysis using a xenograft mouse model was demonstrated. We believe these results will be useful for fabricating molecular MRI probes for the diagnosis of cancer.

Molecular recognition of proteolytic activity in metastatic cancer cells using fluorogenic gold nanoprobes.

We describe the development of biomarker-sensitive nanoprobes based on nanoparticle surface energy transfer (NSET) effect that enabling recognition of the expression of membrane type-1 matrix metalloproteinase (MT1-MMP) anchored on invasive cancer cells and its proteolytic activity simultaneously. First of all, we confirmed invasiveness of cancer cell lines (HT1080 and MCF7) via migration and invasion assay. We also prepared gold nanoparticle (GNP) acts as a quencher for fluorescein isothiocyanate (FITC). This FITC is conjugated in end-terminal of activatable fluorogenic peptide (ActFP). The ActFP attach to surface of GNP (GNP-ActFP) for a targeting moiety and proteolytic activity ligand toward MT1-MMP. The GNP-ActFP can generate fluorescence signal when ActFP is cleaved by proteolytic activity after targeting toward MT1-MMP. In order to study specificity for MT1-MMP, GNP-ActFP is treated to HT1080 and MCF7 cells, and then, we determine the in vitro targeting potential and fluorogenic activity of GNP-ActFP for MT1-MMP via fluorescence multi-reader. We also confirmed fluorogenic activity of GNP-ActFP via confocal microscopic imaging, and finally, endocytosis of GNP-ActFP is observed via cellular transmission electron microscopic imaging.

Protective effect of cyanidin-3-O-ß-D-glucopyranoside fraction from mulberry fruit pigment against oxidative damage in streptozotocin-induced diabetic rat bladder.

AIMS: To determine whether cyanidin-3-O-ß-D-glucopyranoside (C3G) fraction from mulberry fruit pigment has protective effects against bladder dysfunction on streptozotocin-induced diabetic rats METHODS: Sprague-Dawley rats were divided into three groups (n = 12 in each): normal, diabetes (DM), and DM treated with C3G fraction (DM + C3G). The DM and DM + C3G groups received a single injection of streptozotocin (50 mg/kg) intraperitoneally. Four weeks after the induction of diabetes, the DM + C3G group was treated with daily oral C3G (10 mg/kg) dissolved in water, for 8 weeks. After 12 weeks of streptozotocin injections, rats in each group underwent cystometrography and bladders were used for evaluation of apoptosis and oxidative stress. RESULTS: The DM group showed a markedly lower maximal intravesical pressure than that observed in the control group, whereas rats in the DM + C3G group showed improved maximum intravesical pressure associated with minimization of apoptosis, and increased levels of Akt and Bad phosphorylation, implying inhibition of pro-apoptotic stimuli. The level of 8-hydroxy-2-deoxyguanosine, a marker of oxidative stress, was significantly greater in the DM group compared to the control group and it was significantly reduced in the C3G treated group. Immunoblotting revealed a significant decrease in the levels of the superoxide dismutase protein and nerve growth factor in the DM group compared with the control group; however, these proteins were upregulated in the DM + C3G group compared with the DM group. CONCLUSIONS: The study is the first to suggest that C3G fraction have a potency to protect the bladder under conditions of diabetes-induced oxidative stress.