A Possible Path to the RNA World: Enantioselective and Diastereoselective Purification of Ribose

A theoretical mechanism resulting in the prebiotic appearance of enantiopure ribose, which would be needed for the origin of RNA and the “RNA world” is proposed. The mechanism simultaneously explains the emergence of biological homochirality and could answer the question of why nucleic acids are based on ribose rather than another sugar. Cleavage of certain non-chiral mineral crystals is known to lead to formation of chiral surfaces. In a chromatography-like process a mixture of racemic carbohydrates originating from the formose reaction is proposed to have been separated on such a chiral surface. Monosaccharides interact with surfaces through their hydroxyl groups, either by hydrogen bond formation or complex formation with metal ions. α-Ribopyranose, which has all hydroxyl groups on one side of the ring, is known to interact more strongly than other sugars with surfaces, as corroborated by certain chromatographic and electrophoresis data. A similar scenario leading to enantiopure ribose is separation on a flat, but not necessarily chiral surface in the presence of a strong electric field capable of orienting highly polar derivatives of sugars. Portions of this work were presented to Mid-Atlantic Regional Meeting of ACS, Hershey, PA, USA, June 05, 2006.     

Oxidation-sensitive mechanisms,vascular apoptosis and atherosclerosis

Increased generation of oxidants, resulting from disruption of aerobic metabolism and from respiratory burst, is an essential defense mechanism against pathogens and aberrant cells. However, oxidative stress can also trigger and enhance deregulated apoptosis or programmed cell death, characteristic of atherosclerotic lesions. Oxidation-sensitive mechanisms also modulate cellular signaling pathways that regulate vascular expression of cytokines and growth factors, and influence atherogenesis, in particular when increased levels of plasma lipoproteins provide ample substrate for lipid peroxidation and lead to increased formation of adducts with lipoprotein amino acids. In some cases, increased oxidation and apoptosis in a group of cells might be beneficial for survival and function of other groups of arterial cells. However, overall, oxidation and apoptosis appear to promote the progression of diseased arteries towards a lesion that is vulnerable to rupture, and to give rise to myocardial infarction and ischemic stroke. Recent rapid advances in our understanding of the interactions between oxidative stress, apoptosis and arterial gene regulation suggest that selective interventions targeting these biological functions have great therapeutic potential.     

Ribonucleotide reductase activity during amphibian development.

Uniformly labeled [3H] uridine is incorporated into DNA by dissociated Pleurodeles blastulae; the label is found in cytosine and to a much lesser extent in thymine. Ribonucleotide reductase activity cannot be detected in full grown oocytes of Xenopus and Pleurodeles, but is present in unfertilized egg. The enzyme is synthesized (or activated) when maturation is induced in Xenopus oocytes by in vitro hormonal treatment. The enzymatic activity increases after fertilization and reaches a peak at the 2--4 cell stage; it decreases at the blastula, gastrula and neurula stages to the low level initially present in unfertilized eggs. The enzyme is no longer detectable in swimming tadpoles. Addition of hydroxyurea (1 mg/ml) to fertilized eggs leads to complete loss of ribonucleotide reductase activity: cycloheximide (20 mug/ml) inhibits the rise in activity characteristic of early cleavage, while actinomycin D (20 mug/ml) has no effect. The significance of these results in discussed.     

Dickkopf1 regulates fate decision and drives breast cancer stem cells to differentiation: an experimentally supported mathematical model

Background

Modulation of cellular signaling pathways can change the replication/differentiation balance in cancer stem cells (CSCs), thus affecting tumor growth and recurrence. Analysis of a simple, experimentally verified, mathematical model suggests that this balance is maintained by quorum sensing (QS).

Methodology/Principal Findings

To explore the mechanism by which putative QS cellular signals in mammary stem cells (SCs) may regulate SC fate decisions, we developed a multi-scale mathematical model, integrating extra-cellular and intra-cellular signal transduction within the mammary tissue dynamics. Preliminary model analysis of the single cell dynamics indicated that Dickkopf1 (Dkk1), a protein known to negatively regulate the Wnt pathway, can serve as anti-proliferation and pro-maturation signal to the cell. Simulations of the multi-scale tissue model suggested that Dkk1 may be a QS factor, regulating SC density on the level of the whole tissue: relatively low levels of exogenously applied Dkk1 have little effect on SC numbers, whereas high levels drive SCs into differentiation. To verify these model predictions, we treated the MCF-7 cell line and primary breast cancer (BC) cells from 3 patient samples with different concentrations and dosing regimens of Dkk1, and evaluated subsequent formation of mammospheres (MS) and the mammary SC marker CD44⁺CD24^lo. As predicted by the model, low concentrations of Dkk1 had no effect on primary BC cells, or even increased MS formation among MCF-7 cells, whereas high Dkk1 concentrations decreased MS formation among both primary BC cells and MCF-7 cells.

Conclusions/Significance

Our study suggests that Dkk1 treatment may be more robust than other methods for eliminating CSCs, as it challenges a general cellular homeostasis mechanism, namely, fate decision by QS. The study also suggests that low dose Dkk1 administration may be counterproductive; we showed experimentally that in some cases it can stimulate CSC proliferation, although this needs validating in vivo.     

Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in middle eastern families

Background

Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity.

Results

A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population.

Conclusions

Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.     

In vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines

Background

The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system.

Methods

Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S) bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function.

Results and Discussion

Water contact angle (65-70°), as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate) and the other (SH/S-S) at the ω-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution.

Conclusions

To the best of our knowledge, immobilization of a protein by the method presented here, with the resulting high enzymatic activity, has never been reported. There are many potential applications for selective localization of active proteins at patterned surfaces, for example, bioMEMS (MEMS - Micro-Electro-Mechanical Systems. Due to the success of the method, presented here, it was decided to continue a research project of a biosensor by transferring it to a high aspect ratio platform - nanotubes.     

A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes--a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.