Trace Determination of Grouper Nervous Necrosis Virus in Contaminated Larvae and Pond Water Samples Using Label-Free Fiber Optic Nanoplasmonic Biosensor.

We developed a fast (<20 min), label-free fiber optic particle plasmon resonance (FOPPR) immunosensing method to detect nervous necrosis virus (NNV), which often infects high-value economic aquatic species, such as grouper. Using spiked NNV particles in a phosphate buffer as samples, the standard calibration curve obtained was linear (R2 = 0.99) and the limit of detection (LOD) achieved was 2.75 × 104 TCID50/mL, which is superior to that obtained using enzyme-linked immunosorbent assay (ELISA). By using an enhancement method called fiber optic nanogold-linked immunosorbent assay (FONLISA), the LOD can be further improved to <1 TCID50/mL, which is comparable to that found by the conventional qPCR method. Employing the larvae homogenate samples of NNV-infected grouper, the results obtained by the FOPPR biosensor agree with those obtained by the quantitative polymerase chain reaction (qPCR) method. We also examined pond water samples from an infected container in an indoor aquaculture facility. The lowest detectable level of NNV coat protein was found to be 0.17 µg/mL, which is one order lower than the LOD reported by ELISA. Therefore, we demonstrated the potential of the FOPPR biosensor as an outbreak surveillance tool, which is able to give warning indication even when the trend of larvae death toll increment is still not clear.

The Effect of Octapeptide Repeats on Prion Folding and Misfolding.

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.

Piperlongumine Suppresses Proliferation of Human Oral Squamous Cell Carcinoma through Cell Cycle Arrest, Apoptosis and Senescence.

Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells.

Autophagy is deficient and inversely correlated with COX-2 expression in nasal polyps: a novel insight into the inflammation mechanism.

BACKGROUND: Nasal polyposis is characterised by persistent inflammation of the upper airways. Autophagy has been implicated in many chronic inflammatory diseases. Whether autophagy plays a role in nasal polyp (NP) inflammation is completely unknown and deserves investigation. METHODS: LC3 and COX-2 expression, the common autophagy and inflammation indicators, respectively, was analysed by immunoblotting in fresh tissues of NP and control nasal mucosa (NM). Primary cultures of NP-derived fibroblasts (NPDFs) and NMDFs were established for in vitro studies. Autophagy was induced by amino acid starvation and LC3 ectopic overexpression or inhibited by 3-methyladenine in the fibroblasts. Inflammation was induced by IL1-ß and TNF-α. LC3 and COX-2 expression was confirmed in NP specimens by immunohistochemistry. RESULTS: LC3 expression was decreased while COX-2 expression was significantly increased in fresh NP tissues compared with the NM control. In NMDFs and NPDFs, autophagy induction by starvation and LC3 overexpression downregulated COX-2 expression. Conversely, autophagy inhibition by 3-methyladenine enhanced COX-2 expression. However, IL1-ß and TNF-α had no effect on autophagy. Immunohistochemical studies on the NP specimens showed that most displayed low LC3 expression, whereas COX-2 was highly expressed in >50% of the specimens. Examination of two consecutive NP sections from the same tissue blocks revealed a negative correlation between LC3 and COX-2 expression. CONCLUSION: Autophagy is deficient in NP tissues and COX-2 is negatively regulated by autophagy in NP-derived fibroblasts. Since COX-2 is essential for the production of pro-inflammatory mediators, this study might help interpret persistent mucosal inflammation in NP. Attenuation of inflammation by restoring autophagy might be a therapeutic strategy for treating NP.

p22phox confers resistance to cisplatin, by blocking its entry into the nucleus.

Cisplatin (CDDP) is a potent chemotherapeutic agent but resistance to the drug remains a major challenge in cancer treatment. To evaluate the efficacy of CDDP in oral squamous cell carcinoma (OSCC), we found that p22phox was highly expressed in CDDP-resistant OSCC specimens. Knockdown of p22phox sensitized OSCC cell lines to CDDP (P < 0.05). Stable overexpression of p22phox augmented CDDP resistance, as evidenced by the significantly higher IC50 values. This cytoprotective effect was attributed to the abrogation of CDDP-induced apoptosis. Akt phosphorylation was increased in p22phox stable lines. However, blocking PI3K/Akt pathway only partially restored CDDP-induced apoptosis. In addition, the overexpressed p22phox in OSCC cells exhibited cytoplasmic localization with enhanced perinuclear expression, consistent with the localization pattern in OSCC specimens. Remarkably, CDDP entry into the nucleus was severely impaired in p22phox-overexpressing cells (P < 0.001), and cytoplasmically accumulated CDDP was co-localized with overexpressed p22phox. This was supported by decreased CDDP-DNA adduct formation and delayed chk1-p53 signaling activation. Together, overexpression of p22phox sequestered CDDP and caused defective CDDP entry into the nucleus, significantly attenuating CDDP-induced apoptosis. Such diminished apoptosis was further abolished by p22phox-activating PI3K/Akt pathway. Our work has suggested a novel biomarker and insight into the mechanism of CDDP resistance.

Autophagy is deficient in nasal polyps: implications for the pathogenesis of the disease.

BACKGROUND: Nasal polyposis is characterized by persistent inflammation but the pathogenesis is complex and still debatable. Autophagy has been associated with many human health problems including chronic inflammatory airway diseases. Whether autophagy plays a role in nasal polyps and could be a therapeutic target is completely unknown. METHODS: We studied light chain 3 (LC3) protein expression, a common indication of autophagy, in fresh tissue specimens of 5 nasal polyps and 6 normal nasal mucosa by Western blot analysis. The results were also confirmed by immunohistochemistry (IHC) using additional 25 paraffin-embedded nasal tissue sections. Finally the autophagic activity was validated in nasal polyp-derived fibroblasts by evaluating the number of green fluorescent protein (GFP)-labeled LC3 puncta. RESULTS: The expression of LC3 was dramatically decreased in all 5 nasal polyp tissues. In contrast, protein kinase B-mechanistic target of rapamycin (Akt-mTOR) signaling, an established negative regulator of autophagy, was significantly activated in these tissues. Immunohistochemical results further demonstrated a negative correlation between autophagy and nasal polyps (p < 0.05). GFP-LC3 puncta formation, an alternative indicator of autophagy, was also diminished in nasal polyp-derived fibroblasts (p < 0.01). CONCLUSION: Autophagy is deficient presumably due to suppression by high Akt-mTOR activity in nasal polyps, which may provide a molecular basis for future mechanistic study of the disease.

Electricity generation and wastewater treatment of oil refinery in microbial fuel cells using Pseudomonas putida.

Microbial fuel cells (MFCs) represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059), a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm) chemical oxygen demand (COD) could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm² in the third cycle with a maximum current density of 0.015 mA/cm² in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10⁻²% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation.

Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique.

Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

Monocyte chemotactic protein 1 (MCP-1) modulates pro-survival signaling to promote progression of head and neck squamous cell carcinoma.

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) recruits monocytes and macrophages to inflammation sites, and inflammatory infiltration correlates with the progression of head and neck squamous cell carcinoma (HNSCC). This study aims to determine whether MCP-1 expression is related to HNSCC malignancy and patient survival. We also investigated the relationship between MCP-1 expression and the phosphorylation state of the pro-survival pathway factors Akt, ERK, and STAT3. METHODS: Expression of MCP-1 and related proteins in HNSCC cell lines was investigated using western blotting. HNSCC patients (34) without distant metastasis at diagnosis were recruited for tissue specimen evaluation of MCP-1 expression and clinical outcomes. The relationship between MCP-1 expression and survival was evaluated using the Cox proportional hazard model with stepwise selection. RESULTS: High-grade HNSCC cell lines were found to have higher levels of active Akt, ERK, and/or STAT3 than did lower grade cell lines under serum-free condition. OCSL, the most malignant cell line, had the highest level of endogenous MCP-1. Administration of exogenous recombinant MCP-1 increased phosphorylation of Akt, ERK, and STAT3 in a dose- and time-dependent manner and increased cellular resistance to serum starvation. Inhibition of Akt, ERK, or STAT3 reduced cell growth and caused cell death. Long-term survival of HNSCC patients was negatively associated with the histological intensity of MCP-1, implicating MCP-1 as a potential prognostic marker for HNSCC. CONCLUSIONS: These results suggest that overexpressed MCP-1 in cancer cells may promote HNSCC progression through upregulating pro-survival signaling pathways. High cellular MCP-1 expression is related to poor overall survival rate in HNSCC patients.

Areca nut extracts exert different effects in oral cancer cells depending on serum concentration: A clue to the various oral alterations in betel quid chewers.

Betel quid chewing is associated with various pathologic alterations in oral mucosa. However, the molecular mechanism behind so many contradictory alterations remains unclear. Here we aimed to build a model to facilitate the related studies in cultured cells. In our results, areca nut extract (ANE) was found to exert different effects in oral cells depending on the supplemented serum level. ANE strongly induced DNA damage, necrotic ballooning, and inflammatory cytokines under lower serum concentration while might convert to facilitate deregulated growth of serum-supplemented cells via modulating the activity/expression of factors such as E-cadherin and Snail. Despite ANE significantly activated NF-κB, a mediator critical for inflammation, inhibition of NF-κB did not prevent the activation of IL8 promoter. We further discovered Y705-dephosphorylated STAT3 might enhance IL8 transcription. Since necrosis and the inflammatory cytokines could cause massive inflammation, infiltration of interstitial fluid might potentiate cellular resistance against the acute cytotoxicity of ANE and further support the proliferation of transforming cells. Induction of VEGF and angiogenesis under lower serum condition also paved the way for cell growth and subsequent metastasis. Accordingly, we concluded that in correlation with serum infiltration ANE caused particular effects in oral cells and possibly the various clinicopathological alterations in vivo.

Liberation of GPI-anchored prion from phospholipids accelerates amyloidogenic conversion.

Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed.

Areca nut extract induces pyknotic necrosis in serum-starved oral cells via increasing reactive oxygen species and inhibiting GSK3ß: an implication for cytopathic effects in betel quid chewers.

Areca nut has been proven to be correlated with various pathologic alterations in oral cavity. However, the mechanisms for such cytopathic effects are still elusive due mostly to the limitations of cell culture systems. Here we discovered that areca nut extract (ANE) induced production of autophagosome vacuoles in cells cultured with rich medium but induced pyknosis and ballooning, two morphological alterations frequently observed in betel quid chewers, in cells under a serum-free culture condition. Permeability of the serum-starved cells to propidium iodide (PI) confirmed ANE induced novel necrosis with pyknosis (pyknotic necrosis), providing a possible explanation for inflammatory infiltration in chewers' mucosa. In these serum-starved cells, ANE strongly induced reactive oxygen species (ROS), which acted as a key switch for the initiation of pyknotic necrosis. Calcium flux was also involved in the morphological alterations. Besides, inhibition of GSK3ß by SB216763 significantly exacerbated the pyknotic necrosis either induced by ANE or H2O2 in serum-starved cells, suggesting that GSK3ß is a critical regulator for ANE/ROS-mediated pyknotic necrosis. Interestingly, LC3-II transition and PARP cleavage were still detected in the serum-starved cells after ANE treatment, suggesting concurrent activation of apoptotic and autophagic pathways. Finally, insulin could counteract the effect of ANE-induced pyknotic necrosis. Taken together, these data provide a platform for studying ANE-induced cytopathogenesis and the first clinical implication for several pathological alterations, such as ballooning and inflammatory infiltration, in betel quid chewers.

Epigenetic deregulation of the anaplastic lymphoma kinase gene modulates mesenchymal characteristics of oral squamous cell carcinomas.

DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n = 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n = 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs.

Calreticulin, an endoplasmic reticulum-resident protein, is highly expressed and essential for cell proliferation and migration in oral squamous cell carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) has emerged as one of the major malignant tumors of the head and neck cancers. However, the molecular mechanism behind tumorigenesis of OSCC is not fully understood. The aim of this study was to investigate the role of calreticulin (CRT), an endoplasmic reticulum-resident protein, in OSCC cells. MATERIALS AND METHODS: Sixteen paired samples of tumor and non-cancerous matched tissue (NCMT), six OSCC cell lines and normal human oral keratinocytes (NHOKs), and oral tissue microarray were used to reveal the expression of CRT by Western blotting and immunohistochemistry. Later, shRNA-mediated stable knockdown of CRT in OSCC cells was generated. The knockdown cell line was used to analyze cell proliferation, colony formation, anchorage-independent growth and cell migration in vitro. RESULTS: CRT was differentially expressed in fresh tumor samples and six OSCC cell lines but not adjacent NCMTs and NHOKs. In oral tissue microarray, we showed that there was positive CRT staining in the vast majority of tumor cases (99/103), in sharp contrast to that in NCMT cases (29/92) (p<0.001). Stable knockdown of CRT in oral cancer cells resulted in significantly reduced growth rate, colony-forming capacity and anchorage-independent growth. This may be attributed to the induction of G0/G1 cell cycle arrest when CRT was depleted in the cells. Both horizontal and vertical movements of the CRT-knockdown stable line were markedly impaired. The phosphorylation levels of focal adhesion kinase (FAK), paxillin and ERK1/2 and the activity of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) were decreased in the CRT-knockdown cells. These results suggest that CRT can regulate oral cancer cell migration through activation of the FAK signaling pathway accompanied with proteolytic degradation of the extracellular matrix (ECM) by MMP-2 and MMP-9. CONCLUSION: Together, this study has defined a novel biological role for CRT in oral cancer. CRT is a potential biomarker and may contribute to the malignant phenotypes of OSCC cells.

A single-chamber microbial fuel cell without an air cathode.

Microbial fuel cells (MFCs) represent a novel technology for wastewater treatment with electricity production. Electricity generation with simultaneous nitrate reduction in a single-chamber MFC without air cathode was studied, using glucose (1 mM) as the carbon source and nitrate (1 mM) as the final electron acceptor employed by Bacillus subtilis under anaerobic conditions. Increasing current as a function of decreased nitrate concentration and an increase in biomass were observed with a maximum current of 0.4 mA obtained at an external resistance (R(ext)) of 1 KΩ without a platinum catalyst of air cathode. A decreased current with complete nitrate reduction, with further recovery of the current immediately after nitrate addition, indicated the dependence of B. subtilis on nitrate as an electron acceptor to efficiently produce electricity. A power density of 0.0019 mW/cm(2) was achieved at an R(ext) of 220 Ω. Cyclic voltammograms (CV) showed direct electron transfer with the involvement of mediators in the MFC. The low coulombic efficiency (CE) of 11% was mainly attributed to glucose fermentation. These results demonstrated that electricity generation is possible from wastewater containing nitrate, and this represents an alternative technology for the cost-effective and environmentally benign treatment of wastewater.

Reversine, a 2,6-disubstituted purine, as an anti-cancer agent in differentiated and undifferentiated thyroid cancer cells.

PURPOSE: A novel and effective treatment is urgently needed to deal with the current treatment dilemma in incurable differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC). Reversine, a small synthetic purine analogue (2,6-disubstituted purine), has been shown to be effective in tumor suppression. METHODS: We performed in vitro evaluation of anti-tumor effects of reversine on proliferation, cell cycle, and apoptosis in human PDTC, ATC, and follicular thyroid cancer cell lines, respectively. RESULTS: Treatment of these three lines with reversine inhibited proliferation in a time- and dose-dependent manner. G2/M accumulation was demonstrated in cell cycle analysis. Reversine induced apoptosis in PDTC cells with caspase-3 and caspase-8 activation, but not caspase-9. Use of a pan-caspase inhibitor before treatment with reversine attenuated cell death. Reversine also showed in vivo growth inhibitory effects on ATC cells in a xenograft nude mice model. CONCLUSIONS: Data demonstrated that reversine is effective in inhibiting the growth of thyroid cancer cells by cell cycle arrest or apoptosis, especially with the more aggressive ATC and PDTC. Apoptosis was induced by the mitochondria-independent pathway. Reversine is therefore worthy of further investigation in clinical therapeutics.

Arecoline downregulates levels of p21 and p27 through the reactive oxygen species/mTOR complex 1 pathway and may contribute to oral squamous cell carcinoma.

Arecoline, the major alkaloid of areca nut, has been shown to cause strong genotoxicity and is considered a potential carcinogen. However, the detailed mechanism for arecoline-induced carcinogenesis remains obscure. In this study, we noticed that the levels of p21 and p27 increased in two oral squamous cell carcinoma cell lines with high confluence. Furthermore, when treated with arecoline, elevated levels of p21 and p27 could be downregulated through the reactive oxygen species/mTOR complex 1 (ROS/mTORC1) pathway. Although arecoline decreased the activity of mTORC1, the amounts of autophagosome-like vacuoles or type II LC3 remained unchanged, suggesting that the downregulation of p21 and p27 was independent of autophagy-mediated protein destruction. Arecoline also caused DNA damage through ROS, indicating that the reduced levels of p21 and p27 might facilitate G (1) /S transition of the cell cycle and subsequently lead to error-prone DNA replication. In conclusion, these data have provided a possible mechanism for arecoline-induced carcinogenesis in subcytolytic doses in vivo.

Rbms3, an RNA-binding protein, mediates the expression of Ptf1a by binding to its 3'UTR during mouse pancreas development.

The development of the pancreas is a complicated process that is regulated on several levels. Pancreas transcription factor 1, alpha subunit (Ptf1a), also known as p48, is a pancreas-specific basic helix-loop-helix transcription factor that is critical for both exocrine pancreas development and maintenance of acinar cell differentiation. Based on a differential screening assay, we identified Rbms3, a gene encoding a glycine-rich RNA-binding protein, to be specifically expressed in the neural tube and the pancreatic rudiment of e10.5 embryos. The presence of Rbms3 in the early developing pancreas suggests that specific post-transcriptional regulation mechanisms play an important role in controlling pancreas development. In this study, we show that Rbms3 binds to the 3'UTR of Ptf1a mRNA, but not the 3'UTR of Pdx1, which is another pancreatic transcription factor. The ectopic expression of Rbms3 stimulates the translation of a reporter gene carrying the Ptf1a 3'UTR. In addition, when Rbms3 expression is suppressed in the AR42J-B13 pancreatic exocrine cell line, the expression of Ptf1a is also down-regulated. These results suggest that binding of Rbms3 to the 3'UTR of Ptf1a regulates the production of the Ptf1a protein and, thereby, indirectly regulates the expression of the Ptf1a downstream target genes.

Reversine suppresses oral squamous cell carcinoma via cell cycle arrest and concomitantly apoptosis and autophagy.

BACKGROUND: The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated. METHODS: Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence. RESULTS: The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy. CONCLUSIONS: Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.

CCAR1 is required for Ngn3-mediated endocrine differentiation.

Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor that specifies pancreatic endocrine cell fates during pancreas development. It can also initiate a transdifferentiation program when expressed in pancreatic exocrine and ductal cells. However, how Ngn3 initiates a transcriptional cascade to achieve endocrine differentiation is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which is a transcriptional coactivator for nuclear receptors, also interacts with Ngn3. The association between Ngn3 and CCAR1 was verified by pull-down assays and co-immunoprecipitation analyses. Using gene reporter assays, we found that CCAR1 is essential for Ngn3 to activate the expression of the reporter genes containing the NeuroD promoter. Moreover, down-regulation of endogenous CCAR1 in the PANC-1 pancreatic ductal cell line inhibits the transdifferentiation program initiated by Ngn3. CCAR1 is, therefore, a novel partner of Ngn3 in mediating endocrine differentiation.