Combinatorial discovery of thermoresponsive cycloammonium ionic liquids.

This work demonstrated, for the first time, the combinatorial discovery and rational identification of small-molecule cycloammonium-based thermoresponsive ionic liquids that exhibit LCST phase transition and carry attractive Tc values in water.

Automated Bright Field Segmentation of Cells and Vacuoles Using Image Processing Technique.

Understanding the mechanisms and other variants of programmed cell death will help provide deeper insight into various disease processes. Although complex procedures are required to distinguish each type of cell death, the formation of vacuoles is one of the important features in some process of cell death under different conditions. Thus, monitoring and counting the number of vacuoles and the ratio of cells with vacuoles is a commonly used method to indicate and quantify the efficacy of the therapy. Several studies have shown that image processing can provide a quick, convenient and precise mean of performing cell detection. Hence, this study uses an image processing technique to detect and quantify vacuolated cells without the need for dyes. The system both counts the number of vacuolated cells and determines the ratio of cells with vacuoles. The performance of the proposed image processing system was evaluated using 38 images. It has been shown that a strong correlation exists between the automated counts and the manual counts. Furthermore, the absolute percentage errors between automated counts and manual counts for cell detection and vacuolated cell detection using data pooled from all images are 3.61 and 3.33%, respectively. A user-friendly graphical user interface (GUI) is also developed and freely available for download, providing researchers in biomedicine with a more convenient instrument for vacuolization analysis.

Inverse Correlation between Methylation and Expression of the Delta-like Ligand 1 Gene in Gastric Cancer.

Notch signaling is a candidate pathway that transmits environmental information into the cell and interferes with the epigenome of gastric cancer. This study aimed to explore if the Notch pathway was abnormally regulated during gastric tumorigenesis. To achieve the goal, Delta-like ligand 1 (DLL1) gene expression, Notch upstream signal, promoter methylation and its correlation with DLL1 expression were examined by methylation-specific polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in cultured gastric cancer cell lines or gastric cancer patient samples. Immunostainings and tissue arrays (n = 40) were used to confirm the DLL1 expression was down-regulated in cancer cells. Transient or stable Notch1 active domain (NICD)-overexpression suppressed proliferation of the gastric cells but the in vivo tumor growth was enhanced. The results of abnormal DLL1 methylation and expression observed in early gastric lesions and in gastric cancers may be relevant to the pathogenesis of gastric cancer.

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture.

Notch1-promoted TRPA1 expression in erythroleukemic cells suppresses erythroid but enhances megakaryocyte differentiation.

The Notch1 pathway plays important roles in modulating erythroid and megakaryocyte differentiation. To screen the Notch1-related genes that regulate differentiation fate of K562 and HEL cells, the expression of transient receptor potential ankyrin 1 (TRPA1) was induced by Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor. N1IC and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) bound to TRPA1 promoter region to regulate transcription in K562 cells. Transactivation of TRPA1 promoter by N1IC depended on the methylation status of TRPA1 promoter. N1IC and Ets-1 suppressed the DNA methyltransferase 3B (DNMT3B) level in K562 cells. Inhibition of TRPA1 expression after Notch1 knockdown could be attenuated by nanaomycin A, an inhibitor of DNMT3B, in K562 and HEL cells. Functionally, hemin-induced erythroid differentiation could be suppressed by TRPA1, and the reduction of erythroid differentiation of both cells by N1IC and Ets-1 occurred via TRPA1. However, PMA-induced megakaryocyte differentiation could be enhanced by TRPA1, and the surface markers of megakaryocytes could be elevated by nanaomycin A. Megakaryocyte differentiation could be reduced by Notch1 or Ets-1 knockdown and relieved by TRPA1 overexpression. The results suggest that Notch1 and TRPA1 might be critical modulators that control the fate of erythroid and megakaryocyte differentiation.

Analysis of dermal papilla cell interactome using STRING database to profile the ex vivo hair growth inhibition effect of a vinca alkaloid drug, colchicine.

Dermal papillae (DPs) control the formation of hair shafts. In clinical settings, colchicine (CLC) induces patients' hair shedding. Compared to the control, the ex vivo hair fiber elongation of organ cultured vibrissa hair follicles (HFs) declined significantly after seven days of CLC treatment. The cultured DP cells (DPCs) were used as the experimental model to study the influence of CLC on the protein dynamics of DPs. CLC could alter the morphology and down-regulate the expression of alkaline phosphatase (ALP), the marker of DPC activity, and induce IκBα phosphorylation of DPCs. The proteomic results showed that CLC modulated the expression patterns (fold>2) of 24 identified proteins, seven down-regulated and 17 up-regulated. Most of these proteins were presumably associated with protein turnover, metabolism, structure and signal transduction. Protein-protein interactions (PPI) among these proteins, established by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, revealed that they participate in protein metabolic process, translation, and energy production. Furthermore, ubiquitin C (UbC) was predicted to be the controlling hub, suggesting the involvement of ubiquitin-proteasome system in modulating the pathogenic effect of CLC on DPC.

Automated counting of bacterial colonies by image analysis.

Research on microorganisms often involves culturing as a means to determine the survival and proliferation of bacteria. The number of colonies in a culture is counted to calculate the concentration of bacteria in the original broth; however, manual counting can be time-consuming and imprecise. To save time and prevent inconsistencies, this study proposes a fully automated counting system using image processing methods. To accurately estimate the number of viable bacteria in a known volume of suspension, colonies distributing over the whole surface area of a plate, including the central and rim areas of a Petri dish are taken into account. The performance of the proposed system is compared with verified manual counts, as well as with two freely available counting software programs. Comparisons show that the proposed system is an effective method with excellent accuracy with mean value of absolute percentage error of 3.37%. A user-friendly graphical user interface is also developed and freely available for download, providing researchers in biomedicine with a more convenient instrument for the enumeration of bacterial colonies.

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.

Interactomics profiling of the negative regulatory function of carbon monoxide on RANKL-treated RAW 264.7 cells during osteoclastogenesis.

BACKGROUND: During osteoclastogenesis, the maturation of osteoclast (OC) progenitors is stimulated by the receptor activator of nuclear factor-κB ligand (RANKL). Excess OC production plays a critical role in the pathogenesis of inflammatory bone disorders. Conversely, the inhibition of abnormal OC proliferation reduces inflammation-induced bone loss. Low concentrations of carbon monoxide (CO) are known to decrease inflammation and OC-mediated bone erosion but the molecular mechanism is unknown. RESULTS: To obtain insight into the biological function of CO, cultured RANKL-treated RAW 264.7 cells were used in an in vitro experimental model of osteoclastogenesis. The results showed that CO inhibited: 1) tartrate-resistant acid phosphatase (TRAP)-positive cell formation; 2) F-actin ring production; 3) c-fos pathway activation; 4) the expression of cathepsin K, TRAP, calcitonin receptor, and matrix metalloproteinase-9 mRNAs; 5) the expression of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 in translation. Protein-protein interaction analysis predicted mitogen-activated protein kinase kinase kinase 4 as the controlling hub. CONCLUSIONS: Low-concentrations of CO (250 ppm) may inhibit osteoclastogenesis. Data from STRING- and IPA-based interactome analyses suggested that the expression of proteins with the functions of signal transduction, enzymes, and epigenetic regulation are significantly altered by CO during RANKL-induced osteoclastogenesis. Our study provides the first interactome analysis of osteoclastogenesis, the results of which supported the negative regulation of OC differentiation by CO.

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.

Downregulation of connective tissue growth factor by LPS/IFN-γ-induced nitric oxide is reversed by aristolochic acid treatment in glomerular mesangial cells via STAT-1α and NF-κB signaling.

Aristolochic acid (AA) is a common cause of Chinese herb nephropathy. The mechanisms involved in the pathogenesis of AA nephropathy (AAN) are intricate. One well-documented effect of AA in the kidney is its pro-fibrotic activity. Nitric oxide (NO), a messenger gas generated from l-arginine, is the product of nitric oxide synthase (NOS). NO is involved in renal hemodynamics and exerts cytoprotective effects against renal injury. In the present study, the role of NO in AAN was investigated in MES-13 cells, a glomerular mesangial cell line. NO endogenously generated by the induction of inducible nitric oxide synthase (iNOS) with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) significantly downregulated connective tissue growth factor (CTGF) protein expression in MES-13 cells. AA significantly suppressed LPS/IFN-γ-induced NO production and reversed CTGF expression that was downregulated by LPS/IFN-γ. AA decreased iNOS gene and protein expressions in a concentration-dependent manner. AA caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation and interferon response factor-1 (IRF-1) mRNA expression. Furthermore, AA attenuated IκB phosphorylation and reduced NF-κB translocation to the nuclear fraction. Taken together, our data indicate that AA reversed the CTGF expression inhibited by LPS/IFN-γ treatment via suppression of NO and iNOS expressions in MES-13 cells through inhibition of the JAK/STAT-1α and NF-κB signaling pathways. NO potentially exerts antifibrotic activity by down regulation of CTGF in MES-13 cells and inhibition of the iNOS gene by AA might partially account for the fibrotic effects of AA in nephropathy.

Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes.

Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

Curcumin Protects against UVB-Induced Skin Cancers in SKH-1 Hairless Mouse: Analysis of Early Molecular Markers in Carcinogenesis.

Curcumin (CUR) has been shown to possess a preventive effect against various cancers and interfere with multiple-cell signaling pathways. We evaluated the protective effects of CUR in regression of UVB-induced skin tumor formation in SKH-1 hairless mice and its underlying early molecular biomarkers associated with carcinogenesis. Mice irradiated with UVB at 180 mJ/cm(2) twice per week elicited 100% tumor incidence at 20 weeks. Topical application of CUR prior to UVB irradiation caused delay in tumor appearance, multiplicity, and size. Topical application of CUR prior to and immediately after a single UVB irradiation (180 mJ/cm(2)) resulted in a significant decrease in UVB-induced thymine dimer-positive cells, expression of proliferative cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase in p53 and p21/Cip1-positive cell population in epidermis. Simultaneously, CUR also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels. The results suggest that the protective effect of CUR against photocarcinogenesis is accompanied by downregulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-κB, and of inflammatory responses involving COX-2, PGE2, and NO, while upregulation of p53 and p21/Cip1 to prevent DNA damage and facilitate DNA repair.

Activation of the Notch1/STAT3/Twist signaling axis promotes gastric cancer progression.

Gastric carcinoma is one of the most common malignancies and a lethal cancer in the world. Notch signaling and transcription factors STAT3 (signal transducer and activator of transcription 3) and Twist regulate tumor development and are critical regulators of gastric cancer progression. Herein, the relationship among Notch, STAT3 and Twist pathways in the control of gastric cancer progression was studied. We found that Twist and phosphorylated STAT3 levels were promoted by the activated Notch1 receptor in human stomach adenocarcinoma SC-M1, embryonic kidney HEK293 and erythroleukemia K562 cells. Notch1 signaling dramatically induced Twist promoter activity through a C promoter binding factor-1-independent manner and STAT3 phosphorylation. Overexpression of Notch1 receptor intracellular domain (N1IC) enhanced the interaction between nuclear STAT3 and Twist promoter in cells. Gastric cancer progression of SC-M1 cells was promoted by N1IC through STAT3 phosphorylation and Twist expression including colony formation, migration and invasion. STAT3 regulated gastric cancer progression of SC-M1 cells via Twist. N1IC also elevated the progression of other gastric cancer cells such as AGS and KATO III cells through STAT3 and Twist. The N1IC-promoted tumor growth and lung metastasis of SC-M1 cells in mice were suppressed by the STAT3 inhibitor JSI-124 and Twist knockdown. Furthermore, Notch1 and Notch ligand Jagged1 expressions were significantly associated with phosphorylated STAT3 and Twist levels in gastric cancer tissues of patients. Taken together, these results suggest that Notch1/STAT3/Twist signaling axis is involved in progression of human gastric cancer and modulation of this cascade has potential for the targeted combination therapy.

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.

Ionic liquids promote PCR amplification of DNA.

A bicyclic imidazolium ionic liquid (4d), [b-4C-im][Br], was found to be highly effective not only for promoting PCR of GC-rich DNA by minimizing non-specific amplification, but also for facilitating PCR of normal-GC DNA under mild conditions.

Comparative bioelectricity production from various wastewaters in microbial fuel cells using mixed cultures and a pure strain of Shewanella oneidensis.

Current and power density from four wastewaters, agriculture (AWW), domestic (DWW), paper (PWW), and food/dairy (FDWW), were comparatively evaluated in combination with three inocula: wastewater endogenous microbes (MFC1), Shewanella oneidensis MR-1 (MFC2), and wastewater endogenous microbes with MR-1 (MFC3) in single chamber microbial fuel cells (MFC). Using AWW (0.011 mA/cm(2); 0.0013 mW/cm(2)) and DWW (0.017 mA/cm(2); 0.0036 mW/cm(2)), MFC2 was the best candidate providing the maximum current, whereas AWW-MFC1 and DWW-MFC1 were unable to construct a well-established MFC. FDWW produced a maximum current from MFC3 (0.037 mA/cm(2); 0.015 mW/cm(2)), and confirmed the unsuitability of MFC2 at an alkaline pH. FDWW-MFC3 also performed best with the highest substrate degradation and coulombic efficiency. Mixed culture in MFC3 resulted in higher current generation under the influence of MR-1 (except in PWW), indicating the endogenous microbes were not solely responsible for the current but the outperformance was significantly attributed to the association of MR-1.

Notch2-induced COX-2 expression enhancing gastric cancer progression.

Gastric carcinoma is one of the most common and mortal types of malignancy worldwide. To date, the mechanisms controlling its aggressiveness are not yet fully understood. Notch signal pathway can function as either an oncogene or a tumor suppressor in tumorigenesis. Four members (Notch1-4) of Notch receptors were found in mammals and each exhibits distinct roles in tumor progression. Previous study showed that the activated Notch1 receptor promoted gastric cancer progression through cyclooxygenase-2 (COX-2). This study addressed whether Notch2 signal pathway is also involved in gastric cancer progression. Constitutive expression of Notch2 intracellular domain (N2IC), the activated form of Notch2 receptor, promoted both cell proliferation and xenografted tumor growth of human stomach adenocarcinoma SC-M1 cells. The colony formation, migration, invasion, and wound-healing abilities of SC-M1 cells were enhanced by N2IC expression, whereas these abilities were suppressed by Notch2 knockdown. Similarly, Notch2 knockdown inhibited cancer progressions of AGS and AZ521 gastric cancer cells. Expression of N2IC also caused epithelial-mesenchymal transition in SC-M1 cells. Furthermore, N2IC bound to COX-2 promoter and induced COX-2 expression through a CBF1-dependent manner in SC-M1 cells. The ability of N2IC to enhance tumor progression in SC-M1 cells was suppressed by knockdown of COX-2 or treatment with NS-398, a COX-2 inhibitor. Moreover, the suppression of tumor progression by Notch2 knockdown in SC-M1 cells was reversed by exogenous COX-2 or its major enzymatic product PGE(2) . Taken together, this study is the first to demonstrate that the Notch2-COX-2 signaling axis plays an important role in controlling gastric cancer progression.

Removal of mercury by foam fractionation using surfactin, a biosurfactant.

The separation of mercury ions from artificially contaminated water by the foam fractionation process using a biosurfactant (surfactin) and chemical surfactants (SDS and Tween-80) was investigated in this study. Parameters such as surfactant and mercury concentration, pH, foam volume, and digestion time were varied and their effects on the efficiency of mercury removal were investigated. The recovery efficiency of mercury ions was highly sensitive to the concentration of the surfactant. The highest mercury ion recovery by surfactin was obtained using a surfactin concentration of 10 × CMC, while recovery using SDS required < 10 × CMC and Tween-80 >10 × CMC. However, the enrichment of mercury ions in the foam was superior with surfactin, the mercury enrichment value corresponding to the highest metal recovery (10.4%) by surfactin being 1.53. Dilute solutions (2-mg L(-1) Hg(2+)) resulted in better separation (36.4%), while concentrated solutions (100 mg L(-1)) enabled only a 2.3% recovery using surfactin. An increase in the digestion time of the metal solution with surfactin yielded better separation as compared with a freshly-prepared solution, and an increase in the airflow rate increased bubble production, resulting in higher metal recovery but low enrichment. Basic solutions yielded higher mercury separation as compared with acidic solutions due to the precipitation of surfactin under acidic conditions.

Impact of taxol on dermal papilla cells--a proteomics and bioinformatics analysis.

Dermal papilla (DP) cells play a regulatory role in hair growth, and also play a role in alopecia (hair loss). However, effects of taxol, which is a widely used chemotherapy drug, on DP cells remain unclear, despite that theoretically taxol can impact on DP cells to contribute to taxol-induced alopecia. To better understand pathophysiology of taxol-induced damage in DP cells, morphological and biochemical analyses were performed to check whether taxol can cause apoptosis in cultured DP cells or not. If it can, proteomics and bioinformatics analyses were then performed to investigate the protein networks which are impacted by the taxol treatment. Our data showed that taxol can cause apoptotic damage in DP cells in a concentration-dependant manner, as demonstrated by various apoptotic markers. Proteomic analysis on DP cells treated with the lowest apoptosis-inducible concentration of taxol revealed that taxol can affect expression of proteins involved in Ca2+-regulated biological processes, vesicles transport, protein folding, reductive detoxification, and biomolecules metabolism. Furthermore, bioinformatics analysis indicated that taxol can impact on multiple biological networks. Taken together, this biochemical, proteomics, and bioinformatics data may give an insight into pathophysiology of taxol-induced damage in DP cells and shed light on mechanisms underlying taxol-induced alopecia.