Design of low-threshold photonic-crystal surface-emitting lasers with confined gain regions by using selective area intermixing.

Photonic-crystal surface-emitting lasers have many promising properties over traditional semiconductor lasers and are regarded as the next-generation laser sources. However, the minimum achievable lasing threshold of PCSELs is still several times larger than that of VCSELs, and limiting its applications especially if the required power is small. Here, we propose a new design that reduces the gain region in the lateral plane by using selective quantum-well intermixing to reduce the threshold current of PCSELs. By performing theoretical calculations, we confirmed that the threshold current can be lowered by a factor of two to three while keeping the PCSEL's advantage of small divergence angle.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model.

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

A simple and fast alternative method to remove glycerol from chicken semen after cryopreservation.

A concentration of 11% of glycerol is the standard one for sperm cryopreservation in chickens, however, the presence of just 2% glycerol already causes severe fertility reduction, suggesting the necessity of removing glycerol before artificial insemination (AI). The major approach developed for this purpose is serial dilution followed by centrifugation (SDC), which demands special equipment (such as a refrigerate room) to maintain post-thaw semen at 4 °C, besides being time consuming. Therefore, we attempted to develop a simple method to remove glycerol from chicken frozen-thawed semen based on a colloidal gel, Percoll, which is ordinarily used to select motile and viable sperm in mammals as well as in fresh chicken semen. In this study, we used a Percoll based glycerol removal solution (GRS) containing sucrose to avoid frozen-thawed sperm suffering from osmotic stress. Subsequently, several conditions including GRS compositions (GRS A, B, C and D) and centrifugation temperatures (4 and 20 °C) were compared by their influence on sperm in vitro parameters. Afterwards, GRS A and D were selected for fertility evaluation, compared to conventional SDC method. Our results showed that the fertility with GRS A at both 4 and 20 °C were higher than GRS D (p < 0.05) and similar or even superior to the fertility obtained with SDC method. Altogether, our novel GRS protocol is a valuable method for chicken sperm cryobanking policy, supported by its notable results of fertility as well as saving 44% of time, with a simple equipment at flexible operation temperatures of 4 or 20 °C.

The autocrine glycosylated-GREM1 interacts with TGFB1 to suppress TGFß/BMP/SMAD-mediated EMT partially by inhibiting MYL9 transactivation in urinary carcinoma.

PURPOSE: Urothelial carcinoma (UC) is a common disease in developed counties. This study aimed to identify autocrine roles and signaling pathways of gremlin 1, DAN family BMP antagonist (GREM1), which inhibits tumor growth and epithelial-mesenchymal transition (EMT) in UC. METHODS: Systematic in vitro and in vivo studies using genetic engineering, different urinary bladder urothelial carcinoma (UBUC)-derived cell lines, and mouse models were performed, respectively. Further, primary upper tract urothelial carcinoma (UTUC) and UBUC specimens were evaluated by immunohistochemistry. RESULTS: GREM1 protein levels conferred better disease-specific and metastasis-free survival rates and played an independent prognostic factor in UTUC and UBUC. Hypermethylation is the primary cause of low GREM1 levels. In different UBUC-derived cell lines, the autocrine/secreted and glycosylated GREM1 interacted with transforming growth factor beta 1 (TGFB1) and inhibited TGFß/BMP/SMAD signaling and myosin light chain 9 (MYL9) transactivation, subsequently cell proliferation and epithelial-mesenchymal transition (EMT). Secreted and glycosylated GREM1 also suppressed tumor growth, metastasis, and MYL9 levels in the mouse model. Instead, cytosolic GREM1 promoted cell proliferation and EMT by activating the tumor necrosis factor (TNF)/AKT/nuclear factor kappa B (NFκB) axis. CONCLUSIONS: Clinical associations, animal models, and in vitro indications provided solid evidence to show that the epithelial autocrine GREM1 is a novel tumor suppressor in UCs. The glycosylated-GREM1 hampered cell proliferation, migration, invasion, and in vitro angiogenesis through interaction with TGFB1 to inactivate TGFß/BMP/SMAD-mediated EMT in an autocrine manner.

Dopaminergic neurons derived from porcine induced pluripotent stem cell like cells function in the Lanyu pig model of Parkinson's disease.

The induced pluripotent stem cells (iPSCs) are able to differentiate into dopaminergic neurons and execute the therapeutic effects for Parkinson's disease (PD). Here, we established a animal model of PD in Lanyu pigs by injecting 5 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP). Next, the porcine iPSC-like cells (piPSC-like cells) were differentiated into D18 neuronal progenitors (D18 NPs) that were transplanted into the striatum to evaluate their therapeutic effects of PD. We showed that after 8 weeks of cell transplantation, the behavior score was significantly ameliorated and fully recovered at the 14th week of cell transplantation. The number of dopaminergic neurons was also significantly improved at the end of the experiment although the number was still about 50% lower than that in the control group. Our findings suggest that piPSC-like cell-derived D18 NPs exhibit a potential for the treatment of PD in the Lanyu pig model.

Sustained-Release and pH-Adjusted Alginate Microspheres-Encapsulated Doxorubicin Inhibit the Viabilities in Hepatocellular Carcinoma-Derived Cells.

The objective of this study aimed to develop biodegradable calcium alginate microspheres carrying doxorubicin (Dox) at the micrometer-scale for sustained release and the capacity of pH regulatory for transarterial chemoembolization. Ultrasonic atomization and CaCl2 cross-linking technologies were used to prepare the microspheres. A 4-by-5 experiment was first designed to identify imperative parameters. The concentration of CaCl2 and the flow rate of the pump were found to be critical to generate microspheres with a constant volume median diameter (~39 µm) across five groups with different alginate: NaHCO3 ratios using each corresponding flow rate. In each group, the encapsulation efficiency was positively correlated to the Dox-loading %. Fourier-transform infrared spectroscopy showed that NaHCO3 and Dox were step-by-step incorporated into the calcium alginate microspheres successfully. Microspheres containing alginate: NaHCO3 = 1 exhibited rough and porous surfaces, high Young's modulus, and hardness. In each group with the same alginate: NaHCO3 ratio, the swelling rates of microspheres were higher in PBS containing 10% FBS compared to those in PBS alone. Microspheres with relatively high NaHCO3 concentrations in PBS containing 10% FBS maintained better physiological pH and higher accumulated Dox release ratios. In two distinct hepatocellular carcinoma-derived cell lines, treatments with microspheres carrying Dox demonstrated that the cell viabilities decreased in groups with relatively high NaHCO3 ratios in time- and dose-dependent manners. Our results suggested that biodegradable alginate microspheres containing relatively high NaHCO3 concentrations improved the cytotoxicity effects in vitro.

Improvement of output efficiency of p-face up photonic-crystal surface-emitting lasers.

We optimized the p-side emission device configuration of photonic-crystal surface-emitting laser (PCSEL) to facilitate the easier chip process and wafer level testing as well as the feasibility of lasing at shorter wavelength. Typically, in order to obtain uniformly distributed current for larger emission area of PCSELs, laser output is designed through the n-side window due to the low hole mobility and thin p-side cladding layer. However, the substrate as well as the epi-layers have to be isolated before the test of each single die on the wafer, which compromised the advantage of wafer-level test of surface emitters. On the other hand, for lasers with emission photon energy higher than the bandgap energy of GaAs substrate, the power will be entirely attenuated. In this study, the optimized p-side emission by applying the transparent conduction layer on top of the p side contact layer to enhance the current distribution and breaking the symmetry of conventional circle pattern in a unit cell to boost the output efficiency is investigated. Through this approach, a high efficiency p-side up PCSEL platform with lower fabrication cost is developed, which is also applicable for short wavelength PCSELs.

Vertically integrated diffractive gratings on photonic crystal surface emitting lasers.

We designed and fabricated a photonic crystal surface emitting laser (PCSEL) with vertically integrated diffractive optical elements on their top to study the mechanism of static beam steering on a single chip. The deflected output beam by the self-formed periodic ITO cladding layer of the PCSEL can be further steered by changing the grating period and azimuthal angle of the diffractive gratings relative to the photonic crystal. Through the analysis of photonic band structure and lasing characteristics, the periodic ITO structure is coupled to the photonic crystal band, whereas the integrated grating serves the diffractive function only. The findings pave the way for the design of PCSELs enabling single or multiple output beam with varying direction capability. This type of laser is regarded as an ideal light source for various applications, such as light detection and ranging and three-dimensional sensing systems.

Tetrazolium Salt WST-8 as a Novel and Reliable Chromogenic Indicator for the Assessment of Boar Semen Quality.

A tetrazolium salt, 2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium (WST-8), has been used widely to determine cell viability; however, its application in the field of reproduction is still limited due to this assay merely providing information regarding cell viability. The aim of this study was to correlate the WST-8 reduction rate with various sperm quality-related parameters (i.e., sperm viability, motility, progressive motility, acrosome integrity and mitochondria integrity) in order to provide a rapid, reliable and affordable assessment for boar semen quality evaluation. Using different ratios of active/damaged sperm cells, we first validated our sample preparations by standard flow cytometry and computer-assisted sperm analysis. Further analyses demonstrated that the most efficient experimental condition for obtaining a reliable prediction model was when sperm concentration reached 300 × 106 cells/mL with the semen/cell-counting kit-8 (CCK-8®) ratio of 200/10 and incubated time of 20 min. Under this set up, the WST-8 reduction rate (differences on optic density reading value, ΔOD at 450 nm) and sperm parameters were highly correlated (p < 0.01) for all sperm parameters evaluated. In the case of limited semen samples, a minimal semen concentration at 150 × 106 cells/mL with the semen/CCK-8® ratio of 200/20 and incubation time for 30 min could still provide reliable prediction of sperm parameters using the WST-8 assay. Our data provide strong evidence for the first time that the WST-8 assay could be used to evaluate boar semen quality with great potential to be applied to different mammalian species.

A protocol for differential staining of cartilages and ossified bones in fetal and adult mouse skeletons using alcian blue and alizarin red S.

The technique for clearing and staining whole specimens consists of many steps. This study discusses the alcian blue/alizarin red S staining method and aims to provide a useful reference and review for users who intend to do this staining. To specifically address the influences of tissue removal on staining results, the mouse fetuses at embryonic stage E18.5 and adult mice at 12 weeks of age were used in this study. The fetuses were divided into three groups: Group 1 skin, muscle, and viscera removed, Group 2 skin and muscle removed, and Group 3 viscera removed. For successful skeletal staining, it was concluded that (1) skin removal from fetuses was necessary for alcian blue staining but unnecessary for alizarin red S staining, (2) removal of muscle surrounding thorax and neck of fetuses could improve transparency effects, (3) retaining fetal viscera would not significantly affect transparency but might avoid the tissue damage, and (4) complete skin, muscle, and viscera removal were essential for good staining of adult mice. The representative images and detailed staining procedures might be good for researchers presently using alcian blue and alizarin red S staining to differentiate cartilages and ossified bones.

Effect of Passivation Layer on the Thin Film Perovskite Random Lasers.

Novel functionalities of disorder-induced scattering effect in random lasers, attributed to low spatial coherence, draw remarkable attention in high-contrast to superior quality speckle-free imaging applications. This paper demonstrates perovskite-polystyrene (PS)-based random lasing action with robust optical performance at room temperature. Optical characterizations are carried out upon perovskite thin films addition with polystyrene of different mixing concentrations (wt.%). A low threshold lasing operation is achieved with an increasing concentration of polystyrene, accompanying a wavy surface texture with high surface roughness. The rough surface dominating multiple scattering effects leads to enhanced feedback efficiency. Moreover, this study also elucidates efficient fabrication process steps for the development of high quality and durable PS-based random lasers. With the advantages of reduced coherent artifacts and low spatial coherence, speckle free projection images of the USAF (U. S. Air Force MIL-STD-150A standard of 1951) resolution test chart are shown for different PS-based random lasers.

Establishment of an induced pluripotent cell line from Taiwan black silkie chick embryonic fibroblasts for replication-incompetent virus production.

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

Inhibition of the formation of autophagosome but not autolysosome augments ABT-751-induced apoptosis in TP53-deficient Hep-3B cells.

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel antimicrotubule drug, ABT-751, in a tumor protein p53 ( TP53)-deficient hepatocellular carcinoma-derived Hep-3B cells. A series of in vitro assays indicated that ABT-751 caused the disruption of the mitotic spindle structure, collapse of mitochondrial membrane potential, generation of reactive oxygen species, DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Hep-3B cells accompanied by alteration of the expression levels of several DNA damage checkpoint proteins and cell cycle regulators. Subsequently, ABT-751 triggered apoptosis along with markedly upregulated several proapoptotic proteins involving in extrinsic, intrinsic, and caspase-mediated apoptotic pathways. A pan-caspase inhibitor suppressed ABT-751-induced apoptosis. ABT-751 also induced autophagy soon after the occurrence of apoptosis through the suppression of AKT serine/threonine kinase/mechanistic target of rapamycin signaling pathway. Exogenous expression of the TP53 gene significantly incurred both apoptosis and autophagy in Hep-3B cells. Pharmacological inhibition of autophagosome (early autophagy) but not autolysosome (late autophagy) enhanced ABT-751-induced apoptosis in TP53-deficient Hep-3B cells. Our study provided a new strategy to augment ABT-751-induced apoptosis in TP53-deficient cells.

First molecular identification of Vorticella sp. from freshwater shrimps in Tainan, Taiwan.

Freshwater shrimps are the most common crustaceans kept in an aquarium. This study was a survey seeking parasites infecting cultured freshwater atyid shrimps at aquarium stores in Tainan, Taiwan. We observed that atyid shrimps were infested with Vorticella and Scutariella. Scutariella is a common shrimp parasite; thus, we focused on Vorticella infection in the atyid shrimps. Vorticella aequilata-like pop TW, a freshwater peritrich ciliate, was isolated from the atyid shrimps. The morphological characteristics were investigated using live observations. Specimens from the population showed identical arrangement of the infraciliature and identical ITS1-5.8SITS2 region sequences. The zooids are bell-shaped, 40-58 µm wide and 47-70 µm in long in vivo. The food vacuole is variable in shape and is located in the middle of the cell. ITS1-5.8S-ITS2 sequences of Vorticella aequilata-like pop TW did not match any available sequences in GenBank. Phylogenetically, Vorticella aequilata-like pop TW clusters with the other Vorticella within the family Vorticellidae and nests with Vorticella aequilata in the subclade. Above all, the morphological characteristics and molecular analyses show that the investigated Vorticella is a Vorticella aequilata-like species. The phylogenetic analyses of ciliates based on the ITS1-5.8S-ITS2 sequences reveal that the Vorticella genus consists of Vorticella morphospecies and that taxonomic revision of the genus is needed. Morphometric criteria and molecular analysis were used to describe and identify the Vorticella specie and this study presents the first molecular identification analysis of the Vorticella species in the cultured atyid shrimps in Tainan, Taiwan.

Porcine-induced pluripotent stem cell-derived osteoblast-like cells ameliorate trabecular bone mass of osteoporotic rats.

AIM: We created rat models of osteoporosis and verified a novel idea to recover bone mass via local cell transplantation. MATERIALS & METHODS: The rats were treated with ovariectomy, 0.1% calcium diet or 3 mg/kg body weight/day of prednisolone and porcine-induced pluripotent stem cell (piPSC)-derived osteoblast-like cells were transplanted into the medullary cavity of the left femurs. RESULTS: The piPSC-derived osteoblast-like cells exerted therapeutic potential on prednisolone treatment group, which confirmed by improvements in trabecular bone volume (15.93 ± 2.20%), bone surface/volume ratio (27.82 ± 1.40 1/mm), thickness (1.40 ± 0.01 mm), separation (0.99 ± 0.10 mm), number (1.13 ± 0.13 1/mm) and total porosity (84.06 ± 2.20%). CONCLUSION: These results first uncovered therapeutic potential of xenotransplantation with piPSCs for glucocorticoid-induced osteoporosis treatment in the rat models.

Porcine induced pluripotent stem cell-derived osteoblast-like cells prevent glucocorticoid-induced bone loss in Lanyu pigs.

The application of appropriate animal models and techniques for the study of osteoporosis is important. Lanyu pigs, a local miniature breed, have been widely used in various biomedical studies in Taiwan. This study aimed to induce bone loss in Lanyu pigs and to examine whether porcine induced pluripotent stem cell (piPSC)-derived osteoblast-like cells could recover bone mass of tibiae via local cell transplantation. piPSCs were directed to differentiate into osteoblast-like cells using osteogenic medium, and differentiated cells expressed osteogenic markers and phenotypes. Twenty mature female Lanyu pigs were divided into four groups, including control (C, 1% calcium diet), treatment 1 (T1, ovariectomy + 1% calcium diet), treatment 2 (T2, ovariectomy + 0.5% calcium diet), and treatment 3 (T3, ovariectomy + 0.5% calcium diet + 1 mg/kg of prednisolone) and were subjected to bone loss induction for twelve months. Micro-CT images revealed that the lowest trabecular bone parameters, such as trabecular bone volume, thickness, separation, number, and total porosity, were detected in the T3 group. The lowest proportions of cortical bone in the proximal metaphysis, proximal diaphysis, and distal diaphysis were also found in the T3 group. These results indicate that ovariectomy, calcium restriction, and prednisolone administration can be applied to induce proper bone loss in Lanyu pigs. After bone loss induction, pigs were subjected to cell transplantation in the left tibiae and were maintained for another six months. Results showed that transplanted piPSC-derived osteoblast-like cells significantly improved trabecular bone structures at transplanted sites and maintained cortical bone structures in the proximal metaphysis. In conclusion, the therapeutic potential of piPSC-derived osteoblast-like cells was confirmed via cell transplantation in the left tibiae of Lanyu pigs. These findings reveal the therapeutic potential of piPSCs for glucocorticoid-induced bone loss in pig models.

Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance in Porcine Induced Pluripotent Stem Cell.

OBJECTIVES: The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells. MATERIALS AND METHODS: In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. RESULTS: Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas. CONCLUSIONS: The pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

Pathogenic Acanthamoeba castellanii Secretes the Extracellular Aminopeptidase M20/M25/M40 Family Protein to Target Cells for Phagocytosis by Disruption.

Acanthamoeba is free-living protist pathogen capable of causing a blinding keratitis and granulomatous encephalitis. However, the mechanisms of Acanthamoeba pathogenesis are still not clear. Here, our results show that cells co-cultured with pathogenic Acanthamoeba would be spherical and floated, even without contacting the protists. Then, the Acanthamoeba protists would contact and engulf these cells. In order to clarify the contact-independent pathogenesis mechanism in Acanthamoeba, we collected the Acanthamoeba-secreted proteins (Asp) to incubate with cells for identifying the extracellular virulent factors and investigating the cytotoxicity process. The Asps of pathogenic Acanthamoeba express protease activity to reactive Leu amino acid in ECM and induce cell-losing adhesion ability. The M20/M25/M40 superfamily aminopeptidase protein (ACA1_264610), an aminopeptidase be found in Asp, is upregulated after Acanthamoeba and C6 cell co-culturing for 6 h. Pre-treating the Asp with leucine aminopeptidase inhibitor and the specific antibodies of Acanthamoeba M20/M25/M40 superfamily aminopeptidase could reduce the cell damage during Asp and cell co-incubation. These results suggest an important functional role of the Acanthamoeba secreted extracellular aminopeptidases in the Acanthamoeba pathogenesis process. This study provides information regarding clinically pathogenic isolates to target specific molecules and design combined drugs.

Transmembrane and Coiled-Coil Domain 1 Impairs the AKT Signaling Pathway in Urinary Bladder Urothelial Carcinoma: A Characterization of a Tumor Suppressor.

Purpose: Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease in developed countries. Cell-cycle dysregulation resulting in uncontrolled cell proliferation has been associated with UBUC development. This study aimed to explore the roles of TMCO1 in UBUCs.Experimental Design: Data mining, branched DNA assay, immunohistochemistry, xenograft, cell culture, quantitative RT-PCR, immunoblotting, stable and transient transfection, lentivirus production and stable knockdown, cell-cycle, cell viability and proliferation, soft-agar, wound-healing, transwell migration and invasion, coimmunoprecipitation, immunocytochemistry, and AKT serine/threonine kinase (AKT) activity assays and site-directed mutagenesis were used to study TMCO1 involvement in vivo and in vitroResults: Data mining identified that the TMCO1 transcript was downregulated during the progression of UBUCs. In distinct UBUC-derived cell lines, changes in TMCO1 levels altered the cell-cycle distribution, cell viability, cell proliferation, and colony formation and modulated the AKT pathway. TMCO1 recruited the PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) to dephosphorylate pAKT1(serine 473) (S473). Mutagenesis at S60 of the TMCO1 protein released TMCO1-induced cell-cycle arrest and restored the AKT pathway in BFTC905 cells. Stable TMCO1 (wild-type) overexpression suppressed, whereas T33A and S60A mutants recovered, tumor size in xenograft mice.Conclusions: Clinical associations, xenograft mice, and in vitro indications provide solid evidence that the TMCO1 gene is a novel tumor suppressor in UBUCs. TMCO1 dysregulates cell-cycle progression via suppression of the AKT pathway, and S60 of the TMCO1 protein is crucial for its tumor-suppressor roles. Clin Cancer Res; 23(24); 7650-63. ©2017 AACR.

Sonic Hedgehog promotes in vitro oocyte maturation and term development of embryos in Taiwan native goats.

The aim of this study was to investigate the effects of Shh (Sonic Hedgehog) protein on caprine oocyte maturation, early embryo development, and developmental competence after embryo transfer of vitrified-thawed in vitro-produced embryos. Cumulus-oocyte complexes (COCs) derived from abattoir were randomly allocated to the in vitro maturation (IVM) medium supplemented with 0 (Control), 0.125, 0.25, 0.5, or 1.0 µg mL-1 recombinant mouse Shh protein. After IVM, COCs were fertilized with frozen-thawed semen and the presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 medium for 9 days. Our results showed that supplementation of Shh (0.25 or 0.5 µg mL-1) enhanced oocyte maturation as compared with the control group (92.4% and 95.0% vs. 86.2%, P < 0.05), yet the effect could be reversed by the simultaneous addition of cyclopamine (an inhibitor of Shh signaling by direct binding to the essential signal transducer Smo). Subsequently, an improved blastocyst rate (66.3 ± 10.9, P < 0.05) was observed for the embryos derived from the oocytes matured in the presence of 0.5 µg mL-1 Shh compared with the control group (41.4 ± 12.9). Expressions of Shh, SMO and Gli1 were observed in the ovaries, granulosa cells, COCs, cumulus cells, oocytes and oviduct epithelia. Notably, Ptch1 was expressed in nearly all of the aforementioned tissues and cells except cumulus cells. The embryos exhibited a higher survival rates in the Shh-supplemented group (37.5%) compared to those without Shh supplementation (14.8%; P < 0.05) after embryo transfer. This study demonstrated the beneficial effects of Shh supplementation on oocyte maturation and subsequent embryo development both in vitro and in vivo, suggesting a functional existence of Shh signaling during the final stage of folliculogenesis and early embryogenesis in caprine.