The Mechanical Properties of Early Drosophila Embryos Measured by High-Speed Video Microrheology

In early development, Drosophila melanogaster embryos form a syncytium, i.e., multiplying nuclei are not yet separated by cell membranes, but are interconnected by cytoskeletal polymer networks consisting of actin and microtubules. Between division cycles 9 and 13, nuclei and cytoskeleton form a two-dimensional cortical layer. To probe the mechanical properties and dynamics of this self-organizing pre-tissue, we measured shear moduli in the embryo by high-speed video microrheology. We recorded position fluctuations of injected micron-sized fluorescent beads with kHz sampling frequencies and characterized the viscoelasticity of the embryo in different locations. Thermal fluctuations dominated over nonequilibrium activity for frequencies between 0.3 and 1000 Hz. Between the nuclear layer and the yolk, the cytoplasm was homogeneous and viscously dominated, with a viscosity three orders of magnitude higher than that of water. Within the nuclear layer we found an increase of the elastic and viscous moduli consistent with an increased microtubule density. Drug-interference experiments showed that microtubules contribute to the measured viscoelasticity inside the embryo whereas actin only plays a minor role in the regions outside of the actin caps that are closely associated with the nuclei. Measurements at different stages of the nuclear division cycle showed little variation.     

Hepatocyte Growth Factor-induced Asef-IQGAP1 Complex Controls Cytoskeletal Remodeling and Endothelial Barrier

Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.     

Removal of nitrate-N by antibiotic exposed bacterial isolates from constructed wetlands

Nitrate-N-removing bacterial strains were isolated from a constructed wetland soil treated with three ionophoric antibiotics: monensin, salinomycin and narasin. Five isolates were selected after initial screening for nitrogen removing potential. Nucleotide sequence analysis of the 16S rRNA gene showed that these isolates were highly similar to Bacillus, and Pseudomonas species. The isolates were assessed for their ability to grow in the presence of ionophoric antibiotics. All strains were found to withstand these pharmaceuticals. In particular, Bacillus subtilis strain BRAZ2B was found to thrive in the drug-exposed wetland environment, showing higher nitrate removal rate than the uninoculated control. The strains were also assessed for nitrogen removal potential under three different C/N ratios: 4, 8 and 12; optimum removal efficiency was observed at C/N 8 for most isolates.     

3,4-Disubstituted oxazolidin-2-ones as constrained ceramide analogs with anticancer activities

Heterocyclic analogs of ceramide as 3-alkanoyl or benzoyl-4-(1-hydroxy-2-enyl)-oxazolidin-2-ones were designed by binding of primary alcohol and amide in sphinogosine backbone as a carbamate. They were synthesized by addition of acyl halide to the common ring 4-(1-t-butyldimethylsilyloxyhexadec-2-enyl)-oxazolidin-2-one which was elaborated from chiral aziridine-2-carboxylate including stereoselective reduction and ring opening reactions as key steps. Other analogs with different carbon frame at C4 position which is corresponding to the sphingoid backbone were prepared from 3-cyclopentanecarbonyl-4-(1-t-butyldimethylsilyloxybut-2-enyl)-oxazolidin-2-one and straight and cyclic alkenes by cross metathesis. All compounds were tested as antileukemic drugs against human leukemia HL-60 cells. Many of them including propionyl, cyclopentanoyl and p-nitrobenzoyl-4-(1-hydroxyhexadec-2-enyl)-oxazolidin-2-ones showed better antileukemic activities than natural C2-ceramide with good correlation between cell death and DNA fragmentation. There is a drastic change of the activities by the carbon chain lengths at C4 position. Cytotoxicity was induced by caspase activation without significant accumulation of endogenous ceramide concentration or any perturbation of ceramide metabolism.     

OntoVisT: A general purpose Ontological Visualization Tool

Ontologies have emerged as a fast growing research topic in the area of semantic web during last decade. Currently there are 204 ontologies that are available through OBO Foundry and BioPortal. Several excellent tools for navigating the ontological structure are available, however most of them are dedicated to a specific annotation data or integrated with specific analysis applications, and do not offer flexibility in terms of general-purpose usage for ontology exploration. We developed OntoVisT, a web based ontological visualization tool. This application is designed for interactive visualization of any ontological hierarchy for a specific node of interest, up to the chosen level of children and/or ancestor. It takes any ontology file in OBO format as input and generates output as DAG hierarchical graph for the chosen query. To enhance the navigation capabilities of complex networks, we have embedded several features such as search criteria, zoom in/out, center focus, nearest neighbor highlights and mouse hover events. The application has been tested on all 72 data sets available in OBO format through OBO foundry. The results for few of them can be accessed through OntoVisT-Gallery. AVAILABILITY: The database is available for free at http://ccbb.jnu.ac.in/OntoVisT.html.     

A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C?G? repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.