SNP identification in unamplified human genomic DNA with gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the ‘point of care’.     

Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH

Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF₄] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF₆]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF₆] in the presence of 40% t-BuOH with CAL-B at 60 °C.     

Re-evaluation of the function of the F420 dehydrogenase in electron transport of Methanosarcina mazei

Methanosarcina mazei is a methanogenic archaeon that is able to thrive on various substrates and therefore contains a variety of redox-active proteins involved in both cytoplasmic and membrane-bound electron transport. The organism possesses a complex branched respiratory chain that has the ability to utilize different electron donors. In this study, two knockout mutants of the membrane-bound F(420) dehydrogenase (ΔfpoF and ΔfpoA-O) were constructed and analyzed. They exhibited severe growth deficiencies with trimethylamine, but not with acetate, as substrates. In cell lysates of the fpo mutants, the F(420):heterodisulfide oxidoreductase activity was strongly reduced, although soluble F(420) hydrogenase was still present. This led to the conclusion that the predominant part of cellular oxidation of the reduced form of F(420) (F(420)H(2)) in Ms. mazei is performed by F(420) dehydrogenase. Enzyme assays of cytoplasmic fractions revealed that ferredoxin (Fd):F(420) oxidoreductase activity was essentially absent in the ΔfpoF mutant. Subsequently, FpoF was produced in Escherichia coli and purified for further characterization. The purified FpoF protein catalyzed the Fd:F(420) oxidoreductase reaction with high specificity (the K(M) for reduced Fd was 0.5 μM) but with low velocity (V(max) = 225 mU·mg(-1)) and was present in the Ms. mazei cytoplasm in considerable amounts. Consequently, soluble FpoF might participate in electron carrier equilibrium and facilitate survival of the Ms. mazei Δech mutant that lacks the membrane-bound Fd-oxidizing Ech hydrogenase.     

A distinct layer of the medulla integrates sky compass signals in the brain of an insect

Mass migration of desert locusts is a common phenomenon in North Africa and the Middle East but how these insects navigate is still poorly understood. Laboratory studies suggest that locusts are able to exploit the sky polarization pattern as a navigational cue. Like other insects locusts detect polarized light through a specialized dorsal rim area (DRA) of the eye. Polarization signals are transmitted through the optic lobe to the anterior optic tubercle (AOTu) and, finally, to the central complex in the brain. Whereas neurons of the AOTu integrate sky polarization and chromatic cues in a daytime dependent manner, the central complex holds a topographic representation of azimuthal directions suggesting a role as an internal sky compass. To understand further the integration of sky compass cues we studied polarization-sensitive (POL) neurons in the medulla that may be intercalated between DRA photoreceptors and AOTu neurons. Five types of POL-neuron were characterized and four of these in multiple recordings. All neurons had wide arborizations in medulla layer 4 and most, additionally, in the dorsal rim area of the medulla and in the accessory medulla, the presumed circadian clock. The neurons showed type-specific orientational tuning to zenithal polarized light and azimuth tuning to unpolarized green and UV light spots. In contrast to neurons of the AOTu, we found no evidence for color opponency and daytime dependent adjustment of sky compass signals. Therefore, medulla layer 4 is a distinct stage in the integration of sky compass signals that precedes the time-compensated integration of celestial cues in the AOTu.     

Thiamine pyrophosphokinase deficiency in encephalopathic children with defects in the pyruvate oxidation pathway

Thiamine pyrophosphate (TPP) is an essential cofactor of the cytosolic transketolase and of three mitochondrial enzymes involved in the oxidative decarboxylation of either pyruvate, α-ketoglutarate or branched chain amino acids. Thiamine is taken up by specific transporters into the cell and converted to the active TPP by thiamine pyrophosphokinase (TPK) in the cytosol from where it can be transported into mitochondria. Here, we report five individuals from three families presenting with variable degrees of ataxia, psychomotor retardation, progressive dystonia, and lactic acidosis. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate but normal pyruvate dehydrogenase complex activity in the presence of excess TPP. A reduced concentration of TPP was found in the muscle and blood. Mutation analysis of TPK1 uncovered three missense, one splice-site, and one frameshift mutation resulting in decreased TPK protein levels.     

Properties of recombinant <Emphasis Type="Italic">Strep</Emphasis>-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

The first hyperthermophilic d-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of d-arabitol, d-xylitol, d-ribulose, or d-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg²⁺ or K⁺. Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of d-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of d-arabitol to d-ribulose and therefore belongs to the class of d-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product d-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, d-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.     

Effects of siRNA-dependent knock-down of cardiolipin synthase and tafazzin on mitochondria and proliferation of glioma cells

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function, and cell proliferation. Changes in CL are often paralleled by changes in the lipid environment of mitochondria that may contribute to mitochondrial function and proliferation. This study aimed to separate the effects of CL content and CL composition from cellular free fatty acid distribution on bioenergetics and proliferation in C6 glioma cells. To this end, cardiolipin synthase and the CL remodelling enzyme, tafazzin, were knocked-down by siRNA in C6 cells. After 72?h of cultivation, we analysed CL composition by means of LC/MS/MS, distribution of cellular fatty acids by means of gas chromatography, and determined oxygen consumption and proliferation. Knock-down of cardiolipin synthase affected the cellular CL content in the presence of linoleic acid (LA) in the culture medium. Knock-down of tafazzin had no consequence with respect to the pattern of cellular fatty acids but caused a decrease in cell proliferation. It significantly changed the distribution of molecular CL species, increased CL content, decreased oxygen consumption, and decreased cell proliferation when cultured in the presence of linoleic acid (LA). The addition of linoleic acid to the culture medium caused significant changes in the pattern of cellular fatty acids and the composition of molecular CL species. These data suggest that tafazzin is required for efficient bioenergetics and for proliferation of glioma cells. Supplementation of fatty acids can be a powerful tool to direct specific changes in these parameters.