Context based mixture model for cell phase identification in automated fluorescence microscopy

BACKGROUND: Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task. RESULTS: The data is generated from Hela H2B GFP cells imaged during a 2-day period with images acquired 15 minutes apart using an automated time-lapse fluorescence microscopy. The patterns are described with four kinds of features, including twelve general features, Haralick texture features, Zernike moment features, and wavelet features. To generate a new set of features with more discriminate power, the commonly used feature reduction techniques are used, which include Principle Component Analysis (PCA), Linear Discriminant Analysis (LDA), Maximum Margin Criterion (MMC), Stepwise Discriminate Analysis based Feature Selection (SDAFS), and Genetic Algorithm based Feature Selection (GAFS). Then, we propose a Context Based Mixture Model (CBMM) for dealing with the time-series cell sequence information and compare it to other traditional classifiers: Support Vector Machine (SVM), Neural Network (NN), and K-Nearest Neighbor (KNN). Being a standard practice in machine learning, we systematically compare the performance of a number of common feature reduction techniques and classifiers to select an optimal combination of a feature reduction technique and a classifier. A cellular database containing 100 manually labelled subsequence is built for evaluating the performance of the classifiers. The generalization error is estimated using the cross validation technique. The experimental results show that CBMM outperforms all other classifies in identifying prophase and has the best overall performance. CONCLUSION: The application of feature reduction techniques can improve the prediction accuracy significantly. CBMM can effectively utilize the contextual information and has the best overall performance when combined with any of the previously mentioned feature reduction techniques

Use of a porous membrane for gas bubble removal in microfluidic channels: physical mechanisms and design criteria

We demonstrate and explain a simple and efficient way to remove gas bubbles from liquid-filled microchannels, by integrating a hydrophobic porous membrane on top of the microchannel. A prototype chip is manufactured in hard, transparent polymer with the ability to completely filter gas plugs out of a segmented flow at rates up to 7.4 microliter/s per mm2 of membrane area. The device involves a bubble generation section and a gas removal section. In the bubble generation section, a T-junction is used to generate a train of gas plugs into a water stream. These gas plugs are then transported towards the gas removal section, where they slide along a hydrophobic membrane until complete removal. The system has been successfully modeled and four necessary operating criteria have been determined to achieve a complete separation of the gas from the liquid. The first criterion is that the bubble length needs to be larger than the channel diameter. The second criterion is that the gas plug should stay on the membrane for a time sufficient to transport all the gas through the membrane. The third criterion is that the gas plug travel speed should be lower than a critical value: otherwise a stable liquid film between the bubble and the membrane prevents mass transfer. The fourth criterion is that the pressure difference across the membrane should not be larger than the Laplace pressure to prevent water from leaking through the membrane

Model based open-loop posture control of a parallel ankle assessment and rehabilitation robot

Ankle injuries are very common in daily life out of musculoskeletal or neurological reasons. Traditional ankle therapy usually requires cooperative and intensive efforts from therapists and patients. Robot-assisted ankle rehabilitation techniques have been actively researched in the past few decades. However, limitations exist such as inappropriate robot design, limited range of motion (ROM) and torque generation capability, or lack of measurement of the interaction between robots and patients. This paper proposes a novel ankle assessment and rehabilitation robot (AARR) that could perform a three-dimensional robotic training with real-time ankle assessment. This study focuses on the control of the robot for ankle rehabilitation rather than assessment. A preliminary test on a healthy subject was conducted using the AARR with a model based open-loop controller. Results show that the robotic training is continuous although the trajectory tracking accuracy is not high. It is concluded that the robotic design has potential for ankle rehabilitation. This model based open-loop controller provides continuous robotic training and is suitable for low precision tracking requirement. It can also serve advanced control schemes as the forward feedback

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell divisio

Co3O4 Nanocrystals on Graphene as a Synergistic Catalyst for Oxygen Reduction Reaction

Catalysts for oxygen reduction and evolution reactions are at the heart of key renewable energy technologies including fuel cells and water splitting. Despite tremendous efforts, developing oxygen electrode catalysts with high activity at low costs remains a grand challenge. Here, we report a hybrid material of Co3O4 nanocrystals grown on reduced graphene oxide (GO) as a high-performance bi-functional catalyst for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). While Co3O4 or graphene oxide alone has little catalytic activity, their hybrid exhibits an unexpected, surprisingly high ORR activity that is further enhanced by nitrogen-doping of graphene. The Co3O4/N-doped graphene hybrid exhibits similar catalytic activity but superior stability to Pt in alkaline solutions. The same hybrid is also highly active for OER, making it a high performance non-precious metal based bi-catalyst for both ORR and OER. The unusual catalytic activity arises from synergetic chemical coupling effects between Co3O4 and graphene.Comment: published in Nature Material

Targeting the IL-6 Dependent Phenotype Can Identify Novel Therapies for Cholangiocarcinoma

The need for new therapies for cholangiocarcinoma is highlighted by their poor prognosis and refractoriness to chemotherapy. Increased production of Interleukin-6 promotes cholangiocarcinoma growth and contributes to chemoresistance by activating cell survival mechanisms. We sought to identify biologically active compounds capable of ameliorating the phenotypic effects of IL-6 expression and to explore their potential therapeutic use for cholangiocarcinoma.A genomic signature associated with Interleukin-6 expression in Mz-ChA-1 human malignant cholangiocytes was derived. Computational bioinformatics analysis was performed to identify compounds that induced inverse gene changes to the signature. The effect of these compounds on cholangiocarcinoma growth was then experimentally verified in vitro and in vivo. Interactions with other therapeutic agents were evaluated using median effects analysis.A group of structurally related compounds, nitrendipine, nifedipine and felodipine was identified. All three compounds were cytotoxic to Mz-ChA-1 cells with an IC50 for felodipine of 26 µM, nitrendipine, 44 µM and nifedipine, 15 µM. Similar results were observed in KMCH-1, CC-LP-1 and TFK-1 cholangiocarcinoma cell lines. At a fractional effect of 0.5, all three agents were synergistic with either camptothecin or gemcitabine in Mz-ChA-1 cells in vitro. Co-administration of felodipine and gemcitabine decreased the growth of Mz-ChA-1 cell xenografts in nude athymic mice.Computational bioinformatics analysis of phenotype-based genomic expression can be used to identify therapeutic agents. Using this drug discovery approach based on targeting a defined tumor associated phenotype, we identified compounds with the potential for therapeutic use in cholangiocarcinoma

Maternal corticotropin-releasing hormone is associated with LEP DNA methylation at birth and in childhood: an epigenome-wide study in Project Viva

BackgroundCorticotropin-releasing hormone (CRH) plays a central role in regulating the secretion of cortisol which controls a wide range of biological processes. Fetuses overexposed to cortisol have increased risks of disease in later life. DNA methylation may be the underlying association between prenatal cortisol exposure and health effects. We investigated associations between maternal CRH levels and epigenome-wide DNA methylation of cord blood in offsprings and evaluated whether these associations persisted into mid-childhood.MethodsWe investigated mother-child pairs enrolled in the prospective Project Viva pre-birth cohort. We measured DNA methylation in 257 umbilical cord blood samples using the HumanMethylation450 Bead Chip. We tested associations of maternal CRH concentration with cord blood cells DNA methylation, adjusting the model for maternal age at enrollment, education, maternal race/ethnicity, maternal smoking status, pre-pregnancy body mass index, parity, gestational age at delivery, child sex, and cell-type composition in cord blood. We further examined the persistence of associations between maternal CRH levels and DNA methylation in children's blood cells collected at mid-childhood (n = 239, age: 6.7-10.3 years) additionally adjusting for the children's age at blood drawn.ResultsMaternal CRH levels are associated with DNA methylation variability in cord blood cells at 96 individual CpG sites (False Discovery Rate <0.05). Among the 96 CpG sites, we identified 3 CpGs located near the LEP gene. Regional analyses confirmed the association between maternal CRH and DNA methylation near LEP. Moreover, higher maternal CRH levels were associated with higher blood-cell DNA methylation of the promoter region of LEP in mid-childhood (P < 0.05, β = 0.64, SE = 0.30).ConclusionIn our cohort, maternal CRH was associated with DNA methylation levels in newborns at multiple loci, notably in the LEP gene promoter. The association between maternal CRH and LEP DNA methylation levels persisted into mid-childhood

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

BACKGROUND: ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis 'senses' NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs. METHODOLOGY/PRINCIPAL FINDINGS: Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47(phox) subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS. CONCLUSION/SIGNIFICANCE: These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can 'sense' redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs

Positive Feedback Regulation between Phospholipase D and Wnt Signaling Promotes Wnt-Driven Anchorage-Independent Growth of Colorectal Cancer Cells

Aberrant activation of the canonical Wnt/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Phopholipase D (PLD) has been implicated in progression of colorectal carcinoma However, an understanding of the targets and regulation of this important pathway remains incomplete and besides, relationship between Wnt signaling and PLD is not known.Here, we demonstrate that PLD isozymes, PLD1 and PLD2 are direct targets and positive feedback regulators of the Wnt/β-catenin signaling. Wnt3a and Wnt mimetics significantly enhanced the expression of PLDs at a transcriptional level in HCT116 colorectal cancer cells, whereas silencing of β-catenin gene expression or utilization of a dominant negative form of T cell factor-4 (TCF-4) inhibited expression of PLDs. Moreover, both PLD1 and PLD2 were highly induced in colon, liver and stomach tissues of mice after injection of LiCl, a Wnt mimetic. Wnt3a stimulated formation of the β-catenin/TCF complexes to two functional TCF-4-binding elements within the PLD2 promoter as assessed by chromatin immunoprecipitation assay. Suppressing PLD using gene silencing or selective inhibitor blocked the ability of β-catenin to transcriptionally activate PLD and other Wnt target genes by preventing formation of the β-catenin/TCF-4 complex, whereas tactics to elevate intracellular levels of phosphatidic acid, the product of PLD activity, enhanced these effects. Here we show that PLD is necessary for Wnt3a-driven invasion and anchorage-independent growth of colon cancer cells.PLD isozyme acts as a novel transcriptional target and positive feedback regulator of Wnt signaling, and then promotes Wnt-driven anchorage-independent growth of colorectal cancer cells. We propose that therapeutic interventions targeting PLD may confer a clinical benefit in Wnt/β-catenin-driven malignancies

Cellular Reactive Oxygen Species Inhibit MPYS Induction of IFNβ

Many inflammatory diseases, as well as infections, are accompanied by elevation in cellular levels of Reactive Oxygen Species (ROS). Here we report that MPYS, a.k.a. STING, which was recently shown to mediate activation of IFNβ expression during infection, is a ROS sensor. ROS induce intermolecular disulfide bonds formation in MPYS homodimer and inhibit MPYS IFNβ stimulatory activity. Cys-64, -148, -292, -309 and the potential C88xxC91 redox motif in MPYS are indispensable for IFNβ stimulation and IRF3 activation. Thus, our results identify a novel mechanism for ROS regulation of IFNβ stimulation