Identifying common prognostic factors in genomic cancer studies: A novel index for censored outcomes

Background  

With the growing number of public repositories for high-throughput genomic data, it is of great interest to combine the results produced by independent research groups. Such a combination allows the identification of common genomic factors across multiple cancer types and provides new insights into the disease process. In the framework of the proportional hazards model, classical procedures, which consist of ranking genes according to the estimated hazard ratio or the p-value obtained from a test statistic of no association between survival and gene expression level, are not suitable for gene selection across multiple genomic datasets with different sample sizes. We propose a novel index for identifying genes with a common effect across heterogeneous genomic studies designed to remain stable whatever the sample size and which has a straightforward interpretation in terms of the percentage of separability between patients according to their survival times and gene expression measurements.     

Use of ATP bioluminescence measurements for the estimation of biomass during biological humification

Summary The development of the microflora during the humification of grape pulp has been investigated by the determination of ATP using the bioluminescence technique. Several extraction methods were tested including the use of dimethylsulphoxide, trichloroacetic acid, grinding and ultrasonification. Dimethylsulphoxide and ultrasonification for 15 sec appeared to be the most effective. The ATP extract was stabilized when it was mixed with 0.75 mM glycine, 4.4 mM Mg-EDTA, pH 7.5 and frozen. The relative error of the ATP assay by bioluminescence did not exceed 6.5%. This method allowed us to show that at least five distinct reproducible microbial phases exist during grape pulp humification. These results show that the microbial biomass changes noticeably and at distinct times during composting.     

Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated β-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.     

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions

Summary Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.     

Purification and characterization of recombinant human interleukin 4. Biological activities, receptor binding and the generation of monoclonal antibodies.

A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml). A protocol was developed for purification of this protein. Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c. Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51%. Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic. Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined. We have characterized the biological activities of the purified rIL-4. This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells. Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM. We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell. Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa. Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.     

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.