A novel yeast-based tool to detect mutagenic and recombinogenic effects simultaneously

In this work, we describe a new yeast-based assay to allow efficient detection of a comprehensive spectrum of genotoxicity events. The constructed diploid Saccharomyces cerevisiae strain allows the simultaneous monitoring of forward mutations, mitotic recombination events and chromosome loss or non-disjunction by direct selection in an easy and highly reproducible approach. The strain contains a DNA module consisting of a single functional copy of the URA3 gene and the kanMX4 gene inserted at the ADE2 locus on the right arm of chromosome XV. The changes of the genotype within the marker region were primarily selected on 5-fluoroorotic acid (5-FOA) agar plates. Further simple phenotypic tests of the 5-FOA-resistant ura3 clones make it possible to analyze the genetic configuration in detail (e.g. point mutations in URA3, gene conversion, crossing-over and chromosome loss). We demonstrate the successful application of our test system by studying the effects of well-known genotoxic agents (UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, aniline and benomyl). We found that the various agents induced mutations and recombination events with different relative frequencies. The integration of the module has generated a hot spot region of mutation and recombination at the borders of the artificially integrated URA3 kanMX4 cassette, which makes the system more sensitive towards DNA-damaging agents. Unlike other test systems, our S. cerevisiae strain is capable to detect a mutagenic effect caused by aniline.     

Coronin 1B antagonizes cortactin and remodels Arp2/3-containing actin branches in lamellipodia

The dendritic actin network generated by the Arp2/3 complex in lamellipodia underlies formation of protrusions, directional sensing, and migration. While the generation of this network is well studied, the mechanisms regulating network disassembly are poorly understood. We report that Coronin 1B disassembles Arp2/3-containing actin filament branches by inducing Arp2/3 dissociation. This activity is antagonized by Cortactin, a filament branch stabilizer. Consistent with this biochemical competition, depletion of both proteins partially rescues defects in lamellipodial dynamics observed upon depletion of either protein alone. Coronin 1B targets actin branches in a manner that is mutually exclusive with the Arp2/3 complex and alters the branch angle. We conclude that Coronin 1B replaces the Arp2/3 complex at actin filament branches as the dendritic network matures and drives the turnover of branched actin networks.     

Detection of microRNA Expression in Human Peripheral Blood Microvesicles

Background

MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.

Methodology/Principal Findings

Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.

Conclusions

This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.     

Interactions between human and carp (Cyprimus carpio) low density lipoproteins (LDL) and LDL receptors

1. We have compared the concentration and chemical composition of carp and human plasma lipoproteins and studied their interaction with human fibroblast LDL receptors. 2. The main lipoproteins in carp are of high density (HDL) in contrast to low density lipoproteins (LDL) in human. 3. Carp lipoproteins are devoid of apolipoprotein (apo) E, a major ligand for interaction with LDL receptors in mammals. 4. Carp very low density lipoproteins (VLDL) and LDL but not HDL nor apoA-I cross react with human LDL in their interaction with LDL receptors on human cultured fibroblasts. 5. Carp liver membranes possess high affinity receptors that are saturable and have calcium dependent ligand specificity (apoB and apoE) similar to human LDL receptor. Carp VLDL and LDL but not HDL nor its major apolipoprotein complexed to L-alpha-phosphatidylcholine dimyristoyl (apoA-I-DMPC) competed with the specific binding of human LDL to this receptor.     

SEPTAL PORES IN THE HETEROBASIDIOMYCETIDAE,PUCCINIA GRAMINIS AND P. RECONDITA

The septal pores in uredial mycelium of Puccinia graminis and P. recondita lack the septal swelling and septal pore cap (dolipore-parenthosome configuration) typically associated with the pores of previously investigated Homobasidiomycetidae and the Tremellales among the Heterobasidiomycetidae. The pores in young hyphae of these two species of Puccinia are characterized by the presence of a cytoplasmic matrix which apparently occludes the pore and acts as a plug, thus preventing the migration of organelles from cell to cell. Large vesicles are typically present at the periphery of the pore matrix and the matrix may be very incompletely bounded by a membrane. Nuclei and other cytoplasmic structures migrate from cell to cell through an opening in the septum lateral to the pore. The available evidence indicates that this peripheral gap in the septum results from a breakdown of a portion of an initially complete septum rather than from incomplete septum formation. In addition to the centripetally formed septa, the hyphae of P. graminis and P. recondita are further compartmentalized by shallow infoldings of the lateral wall and limited unilateral septum formation. There is apparent free passage of cellular material between adjacent compartments.     

OpenAWSEM with Open3SPN2: A fast,flexible, and accessible framework for large-scale coarse-grained biomolecular simulations

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.