Knockdown of Maged1 inhibits cell cycle progression and causes cell death in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are characterized by self-renewal and pluripotency and can undergo differentiation into the three germ layers (ectoderm, mesoderm, and endoderm). Melanoma-associated antigen D1 (Maged1), which is expressed in all developing and adult tissues, modulates tissue regeneration and development. In the present study, we examined the expression and function of Maged1 in mESCs. Maged1 protein and mRNA expression increased during mESC differentiation. The pluripotency of mESCs was significantly reduced through extracellular signal-regulated kinase 1/2 phosphorylation upon knockdown of Maged1, and through G1 cell cycle arrest during cell division, resulting in significantly reduced mESC proliferation. Moreover, the diameter of the embryoid bodies was significantly reduced, accompanied by increased levels of ectodermal differentiation markers and decreased levels of mesodermal and endodermal differentiation markers. Maged1-knockdown mESC lines showed significantly reduced teratoma volumes and inhibition of teratoma formation in nude mice. Additionally, we observed increased ectodermal markers but decreased mesodermal and endodermal markers in teratoma tissues. These findings show that Maged1 affects mESC pluripotency, proliferation, cell cycle, and differentiation, thereby contributing to our understanding of the basic molecular biological mechanisms and potential roles of Maged1 as a regulator of various mESC properties.

Anticancer effects of veratramine via the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin and its downstream signaling pathways in human glioblastoma cell lines.

AIMS: Antitumor effects of veratramine in prostate and liver cancers has been investigated, but it is still unclear whether veratramine can be used as an effective therapeutic agent for glioma. The aim of this study was to evaluate the potential pharmacological mechanism of veratramine in glioma. MAIN METHODS: Using four types of human glioblastoma cell lines, including A172, HS-683, T98G, and U-373-MG the dose-dependent antitumor effect of veratramine was evaluated. The cytotoxicity and cell proliferation were examined by CCK-8, and cell proliferation was further confirmed by anchorage-independent colony formation assay. The cell cycle distribution and apoptotic rate was assessed by flow cytometry, and apoptosis was further evaluated by apoptosis assay. The migration and invasiveness capacity were analyzed by using transwell. Protein and mRNA levels of related factors were determined by western blotting and RT-qPCR, respectively. KEY FINDINGS: Veratramine markedly induced apoptosis, suppressed the cell proliferation via the cell cycle G0/G1 phase arrest, and reduced the capacity for the migration and invasion in human glioblastoma multiforme cell lines. Moreover, veratramine was sufficient to affect the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin signaling pathway and its downstream Mdm2/p53/p21 pathway in human glioblastoma cell lines. SIGNIFICANCE: Antitumor effects of veratramine in suppression of glioma progression was mediated by the regulation of PI3K/Akt/mTOR and Mdm2/p53/p21 signaling pathway.

ZNF507 affects TGF-ß signaling via TGFBR1 and MAP3K8 activation in the progression of prostate cancer to an aggressive state.

BACKGROUND: The progression of prostate cancer (PC) to the highly aggressive metastatic castration-resistant prostate cancer (mCRPC) or neuroendocrine prostate cancer (NEPC) is a fatal condition and the underlying molecular mechanisms are poorly understood. Here, we identified the novel transcriptional factor ZNF507 as a key mediator in the progression of PC to an aggressive state. METHODS: We analyzed ZNF507 expression in the data from various human PC database and high-grade PC patient samples. By establishment of ZNF507 knockdown and overexpression human PC cell lines, we assessed in vitro PC phenotype changes including cell proliferation, survival, migration and invasion. By performing microarray with ZNF507 knockdown PC cells, we profiled the gene clusters affected by ZNF507 knockdown. Moreover, ZNF507 regulated key signal was evaluated by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Finally, we performed xenograft and in vivo metastasis assay to confirm the effect of ZNF507 knockdown in PC cells. RESULTS: We found that ZNF507 expression was increased, particularly in the highly graded PC. ZNF507 was also found to be associated with metastatic PC of a high grade. Loss- or gain-of-function-based analysis revealed that ZNF507 promotes the growth, survival, proliferation, and metastatic properties of PC (e.g., epithelial-mesenchymal transition) by upregulating TGF-ß signaling. Profiling of gene clusters affected by ZNF507 knockdown revealed that ZNF507 positively regulated the transcription of TGFBR1, MAP3K8, and FURIN, which in turn promoted the progression of PC to highly metastatic and aggressive state. CONCLUSIONS: Our findings suggest that ZNF507 is a novel key regulator of TGF-ß signaling in the progression of malignant PC and could be a promising target for studying the development of advanced metastatic PCs.

Three-compartment-breast (3CB) prior-guided diffuse optical tomography based on dual-energy digital breast tomosynthesis (DBT).

Diffuse optical tomography (DOT) is a non-invasive functional imaging modality that uses near-infrared (NIR) light to measure the oxygenation state and the concentration of hemoglobin. By complementarily using DOT with other anatomical imaging modalities, physicians can diagnose more accurately through additional functional image information. In breast imaging, diagnosis of dense breasts is often challenging because the bulky fibrous tissues may hinder the correct tumor characterization. In this work, we proposed a three-compartment-breast (3CB) decomposition-based prior-guided optical tomography for enhancing DOT image quality. We conjectured that the 3CB prior would lead to improvement of the spatial resolution and also of the contrast of the reconstructed tumor image, particularly for the dense breasts. We conducted a Monte-Carlo simulation to acquire dual-energy X-ray projections of a realistic 3D numerical breast phantom and performed digital breast tomosynthesis (DBT) for setting up a 3CB model. The 3CB prior was then used as a structural guide in DOT image reconstruction. The proposed method resulted in the higher spatial resolution of the recovered tumor even when the tumor is surrounded by the fibroglandular tissues compared with the typical two-composition-prior method or the standard Tikhonov regularization method.

IFN-γ Licensing Does Not Enhance the Reduced Immunomodulatory Potential and Migratory Ability of Differentiation-Induced Porcine Bone Marrow-Derived Mesenchymal Stem Cells in an In Vitro Xenogeneic Application.

IFN-γ licensing to mesenchymal stem cells (MSCs) is applied to enhance the therapeutic potential of MSCs. However, although the features of MSCs are affected by several stimuli, little information is available on changes to the therapeutic potential of IFN-γ-licensed differentiated MSCs during xenogeneic applications. Therefore, the present study is aimed at clarifying the effects of adipogenic/osteogenic differentiation and IFN-γ licensing on the in vitro immunomodulatory and migratory properties of porcine bone marrow-derived MSCs in xenogeneic applications using human peripheral blood mononuclear cells (PBMCs). IFN-γ licensing in differentiated MSCs lowered lineage-specific gene expression but did not affect MSC-specific cell surface molecules. Although indoleamine 2,3 deoxygenase (IDO) activity and expression were increased after IFN-γ licensing in undifferentiated MSCs, they were reduced after differentiation. IFN-γ licensing to differentiated MSCs elevated the reduced IDO expression in differentiated MSCs; however, the increase was not sufficient to reach to the level achieved by undifferentiated MSCs. During a mixed lymphocyte reaction with quantification of TNF-α concentration, proliferation and activation of xenogeneic PBMCs were suppressed by undifferentiated MSCs but inhibited to a lesser extent by differentiated MSCs. IFN-γ licensing increasingly suppressed proliferation of PBMCs in undifferentiated MSCs but it was incapable of elevating the reduced immunosuppressive ability of differentiated MSCs. Migratory ability through a scratch assay and gene expression study was reduced in differentiated MSCs than their undifferentiated counterparts; IFN-γ licensing was unable to enhance the reduced migratory ability in differentiated MSCs. Similar results were found in a Transwell system with differentiated MSCs in the upper chamber toward xenogeneic PBMCs in the lower chamber, despite IFN-γ licensing increased the migratory ability of undifferentiated MSCs. Overall, IFN-γ licensing did not enhance the reduced immunomodulatory and migratory properties of differentiated MSCs in a xenogeneic application. This study provides a better understanding of the ways in which MSC therapy can be applied.

Senescence Marker Protein 30 (SMP30): A Novel Pan-Species Diagnostic Marker for the Histopathological Diagnosis of Breast Cancer in Humans and Animals.

Senescence marker protein 30 (SMP30) is a cell survival factor playing an important role in vitamin C synthesis and antiapoptosis. Moreover, its cytoprotective role suggests a possibility to be related to cancer cell survival. Mammary carcinoma is a common cancer in both humans and animals. Because of its histopathological diversity, especially in the early stage, histopathological diagnosis may be complicated; therefore, a diagnostic marker is helpful for confirmation. The present study analyzed the expression pattern of SMP30 in mammary carcinoma in humans, dogs, and cats. Immunohistochemistry, immunofluorescence, and western blot analysis were used to investigate SMP30 expression patterns. The expression was specifically observed in neoplastic glandular epithelial cells. The expression increased with the malignancy of glandular epithelial cells with a highly proliferative status. However, SMP30 expression was low in normal mammary gland tissues or well-differentiated adenoma tissues. The patterns were consistently reproduced in canine primary mammary carcinoma cells and MCF-7 and MDA-MB-231 human carcinoma cell lines. This study provides useful information to understand SMP30 expression in various stages of mammary carcinoma and to suggest its utility as a pan-species diagnostic marker, thereby helping to establish strategies for diagnosing mammary carcinoma in several species.

Feline-type cystic basal cell tumor filled with abundant melanin pigment-rich fluid in a dog.

A 2-year-old castrated male mongrel dog presented with a well-demarcated fluctuant dermal mass, located on the back of the neck. Grossly along with cystic structures filled with a black greasy fluid, when cut open. Microscopically, the mass was multi-lobulated. The lobules consisted of neoplastic basaloid cells and showed central degeneration, forming multiple central cystic structures filled with dark melanin-pigmented materials. Immunohistochemically, the neoplastic cells were strongly positive for CK14 and partially positive for CK19, but negative for CK7, CK8/18, CD34, S-100, Melan-A and α-SMA. Based on the findings, the present case was diagnosed as a feline-type basal cell tumor characterized by cystic structures filled with abundant black fluid.

Congenital lobar emphysema concurrent with pneumothorax and pneumomediastinum in a dog.

A two-year-old castrated male Pomeranian dog was referred with the chief complaints of coughing and subcutaneous emphysema. On physical examination, the crepitant areas were palpable. When auscultated, the right chest was absent of respiratory sound, while the sound of the opposite side was enhanced. Radiographs presented pneumothorax and pneumomediastinum. On computed tomography, hypoattenuated bulla-like lesion at right middle lung lobe and trapped air in mediastinum were shown. After patient stabilization, surgery for excision of affected lobe was performed. During follow-up period, there were no recurrence and complication on radiographic examination. Based on clinical and pathological findings, the dog was diagnosed as congenital lobar emphysema.

Adipose-derived mesenchymal stem cells and platelet-rich plasma synergistically ameliorate the surgical-induced osteoarthritis in Beagle dogs.

BACKGROUND: The purpose of this study is to investigate the clinical effects of platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cell (MSC) as the fundamental treatment of osteoarthritis (OA). METHODS: Twenty four Beagle dogs were used as cranial cruciate ligament transection models. The dogs were divided into four groups (n = 6) according to the intra-articular injection materials: the control group with phosphate-buffered saline (PBS), the PRP group with PRP, the MSC group with MSCs emerged in PBS, and the MSC and PRP co-treatment (MP) group with MSCs and PRP. RESULTS: Lameness score, focal compression strength, articular extracellular matrix (ECM) compositions, histopathology, and real-time PCR were used to evaluate the effects of PRP and MSCs on canine OA. In the order of MP, PRP, and MSC group, these all showed positive effects on the evaluated categories. The lameness scores were lower, and the focal compression strengths of the affected femoral articular surface cartilages were higher than those in the OA control group. Also, the inflammatory changes, when evaluated with Mankin scoring and histomorphologic examination, were significantly ameliorated with the treatment of PRP and/or MSCs. The glycosaminoglycan and collagen composition of extracellular matrix was more favorable in the test groups. The ECM-related genes significantly increased through the up-regulation, while the protein expressions of inflammatory cytokines were decreased through the inhibitory effects of PRP and MSCs on chondrocyte apoptosis and inflammatory cytokines. CONCLUSIONS: Taken together, this study suggests that PRP and MSCs treatments have a beneficial effect on OA via the stimulation of ECM synthesis and chondrocyte proliferation and via the inhibition of inflammatory reaction.

Proteomic Insights into Sulfur Metabolism in the Hydrogen-Producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H2 when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H2-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (≥1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe-S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H2-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.