Effect of Temperature Pre-Exposure on the Locomotion and Chemotaxis of C. elegans

The effect of temperature pre-exposure on locomotion and chemotaxis of the soil-dwelling nematode Caenorhabditis elegans has been extensively studied. The behavior of C. elegans was quantified using a simple harmonic curvature-based model. Animals showed increased levels of activity, compared to control worms, immediately after pre-exposure to 30°C. This high level of activity in C. elegans translated into frequent turns by making ‘complex’ shapes, higher velocity of locomotion, and higher chemotaxis index () in presence of a gradient of chemoattractant. The effect of pre-exposure was observed to be persistent for about 20 minutes after which the behavior (including velocity and ) appeared to be comparable to that of control animals (maintained at 20°C). Surprisingly, after 30 minutes of recovery, the behavior of C. elegans continued to deteriorate further below that of control worms with a drastic reduction in the curvature of the worms'' body. A majority of these worms also showed negative chemotaxis index indicating a loss in their chemotaxis ability.     

Constitutively active GPR6 is located in the intracellular compartments

We used multiple imaging assays to test the hypothesis that GPR6, a constitutively active Gs-coupled receptor, is present on the cell surface. A pHluorin tag at the N-terminus of rat GPR6 expressed in human embryonic kidney 293 (HEK293) cells was not accessible to protons, chymotrypsin or anti-green fluorescent protein antibody, demonstrating that GPR6 is primarily located in intracellular compartments. Similar intracellular localization of pHluorin-tagged GPR6 was found in striatal neurons, where endogenous GPR6 is expressed. Confirmation of Gs-mediated constitutive activity in HEK293 cells and striatal neurons led us to conclude that GPR6 can signal from intracellular compartments.     

Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.     

Nucleotide sequence analysis of DNA. X. Complete nucleotide sequence of the cohesive ends of bacteriophage phi80 DNA

The base sequence of the cohesive ends of bacteriophage φ80 DNA has been shown to be identical to the base sequence of the cohesive ends of bacteriophage lambda DNA.     

A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.     

Alzheimer Aβ Peptide Induces Chromosome Mis-Segregation and Aneuploidy,Including Trisomy 21: Requirement for Tau and APP

Both sporadic and familial Alzheimer''s disease (AD) patients exhibit increased chromosome aneuploidy, particularly trisomy 21, in neurons and other cells. Significantly, trisomy 21/Down syndrome patients develop early onset AD pathology. We investigated the mechanism underlying mosaic chromosome aneuploidy in AD and report that FAD mutations in the Alzheimer Amyloid Precursor Protein gene, APP, induce chromosome mis-segregation and aneuploidy in transgenic mice and in transfected cells. Furthermore, adding synthetic Aβ peptide, the pathogenic product of APP, to cultured cells causes rapid and robust chromosome mis-segregation leading to aneuploid, including trisomy 21, daughters, which is prevented by LiCl addition or Ca²⁺ chelation and is replicated in tau KO cells, implicating GSK-3β, calpain, and Tau-dependent microtubule transport in the aneugenic activity of Aβ. Furthermore, APP KO cells are resistant to the aneugenic activity of Aβ, as they have been shown previously to be resistant to Aβ-induced tau phosphorylation and cell toxicity. These results indicate that Aβ-induced microtubule dysfunction leads to aneuploid neurons and may thereby contribute to the pathogenesis of AD.     

Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues

The movement of proteins within cells can provide dynamic indications of cell signaling and cell polarity, but methods are needed to track and quantify subcellular protein movement within tissue environments. Here we present a semiautomated approach to quantify subcellular protein location for hundreds of migrating cells within intact living tissue using retrovirally expressed fluorescent fusion proteins and time-lapse two-photon microscopy of intact thymic lobes. We have validated the method using GFP-PKCζ, a marker for cell polarity, and LAT-GFP, a marker for T-cell receptor signaling, and have related the asymmetric distribution of these proteins to the direction and speed of cell migration. These approaches could be readily adapted to other fluorescent fusion proteins, tissues and biological questions.     

Meta-analysis of Dense Genecentric Association Studies Reveals Common and Uncommon Variants Associated with Height

Height is a classic complex trait with common variants in a growing list of genes known to contribute to the phenotype. Using a genecentric genotyping array targeted toward cardiovascular-related loci, comprising 49,320 SNPs across approximately 2000 loci, we evaluated the association of common and uncommon SNPs with adult height in 114,223 individuals from 47 studies and six ethnicities. A total of 64 loci contained a SNP associated with height at array-wide significance (p < 2.4 × 10⁻⁶), with 42 loci surpassing the conventional genome-wide significance threshold (p < 5 × 10⁻⁸). Common variants with minor allele frequencies greater than 5% were observed to be associated with height in 37 previously reported loci. In individuals of European ancestry, uncommon SNPs in IL11 and SMAD3, which would not be genotyped with the use of standard genome-wide genotyping arrays, were strongly associated with height (p < 3 × 10⁻¹¹). Conditional analysis within associated regions revealed five additional variants associated with height independent of lead SNPs within the locus, suggesting allelic heterogeneity. Although underpowered to replicate findings from individuals of European ancestry, the direction of effect of associated variants was largely consistent in African American, South Asian, and Hispanic populations. Overall, we show that dense coverage of genes for uncommon SNPs, coupled with large-scale meta-analysis, can successfully identify additional variants associated with a common complex trait.     

Ionic liquid pretreatment of cellulosic biomass: enzymatic hydrolysis and ionic liquid recycle

Ionic liquids (ILs) are promising solvents for the pretreatment of biomass as certain ILs are able to completely solubilize lignocellulose. The cellulose can readily be precipitated with an anti-solvent for further hydrolysis to glucose, but the anti-solvent must be removed for the IL to be recovered and recycled. We describe the use of aqueous kosmotropic salt solutions to form a three-phase system that precipitates the biomass, forming IL-rich and salt-rich phases. The phase behavior of [Emim][Ac] and aqueous phosphate salt systems is presented, together with a process for recycling the [Emim][Ac] and enzymatically hydrolyzing the cellulose. This process reduces the amount of water to be evaporated from recycled IL, permitting efficient recycle of the IL. Material balances on the process, with multiple recycles of the [Emim][Ac], quantify the major components from a Miscanthus feedstock through the pretreatment, separation, and enzymatic hydrolysis steps. A more rapid and higher yielding conversion of cellulose to glucose is obtained by use of the three-phase system as compared to the cellulose obtained from biomass pretreated with IL and precipitated with water. The addition of a kosmotropic salt during the precipitation results in partial delignification of the biomass, which makes the substrate more accessible, enhancing the enzymatic hydrolysis.