A new method for class prediction based on signed-rank algorithms applied to Affymetrix<Superscript>®</Superscript> microarray experiments

Background

The huge amount of data generated by DNA chips is a powerful basis to classify various pathologies. However, constant evolution of microarray technology makes it difficult to mix data from different chip types for class prediction of limited sample populations. Affymetrix^® technology provides both a quantitative fluorescence signal and a decision (detection call: absent or present) based on signed-rank algorithms applied to several hybridization repeats of each gene, with a per-chip normalization. We developed a new prediction method for class belonging based on the detection call only from recent Affymetrix chip type. Biological data were obtained by hybridization on U133A, U133B and U133Plus 2.0 microarrays of purified normal B cells and cells from three independent groups of multiple myeloma (MM) patients.

Results

After a call-based data reduction step to filter out non class-discriminative probe sets, the gene list obtained was reduced to a predictor with correction for multiple testing by iterative deletion of probe sets that sequentially improve inter-class comparisons and their significance. The error rate of the method was determined using leave-one-out and 5-fold cross-validation. It was successfully applied to (i) determine a sex predictor with the normal donor group classifying gender with no error in all patient groups except for male MM samples with a Y chromosome deletion, (ii) predict the immunoglobulin light and heavy chains expressed by the malignant myeloma clones of the validation group and (iii) predict sex, light and heavy chain nature for every new patient. Finally, this method was shown powerful when compared to the popular classification method Prediction Analysis of Microarray (PAM).

Conclusion

This normalization-free method is routinely used for quality control and correction of collection errors in patient reports to clinicians. It can be easily extended to multiple class prediction suitable with clinical groups, and looks particularly promising through international cooperative projects like the "Microarray Quality Control project of US FDA" MAQC as a predictive classifier for diagnostic, prognostic and response to treatment. Finally, it can be used as a powerful tool to mine published data generated on Affymetrix systems and more generally classify samples with binary feature values.

     

Ajuba: a new microtubule-associated protein that interacts with BUBR1 and Aurora B at kinetochores in metaphase

Background information. The role of the LIM‐domain‐containing protein Ajuba was initially described in cell adhesion and migration processes and recently in mitosis as an activator of the Aurora A kinase. Results. In the present study, we show that Ajuba localizes to centrosomes and kinetochores during mitosis. This localization is microtubule‐dependent and Ajuba binds microtubules in vitro. A microtubule regrowth assay showed that Ajuba follows nascent microtubules from centrosomes to kinetochores. Owing to its contribution to mitotic commitment and its microtubule‐dependent localization, Ajuba could also play a role during the metaphase—anaphase transition. We show that Ajuba interacts with Aurora B and BUBR1 [BUB (budding uninhibited by benomyl)‐related 1], two major components of the mitotic checkpoint. Inhibition of BUBR1 by siRNA (small interfering RNA) disrupts chromosome alignment at the metaphase plate and modifies Ajuba localization due to premature mitotic exit. Conclusions. Ajuba is a microtubule‐associated protein that collaborates with Aurora B and BUBR1 at the metaphase—anaphase transition and this may be important to ensure proper chromosome segregation.     

Heparan sulfate mimicry: a synthetic glycoconjugate that recognizes the heparin binding domain of interferon-gamma inhibits the cytokine activity

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-gamma (IFNgamma), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNgamma monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNgamma. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Angstroms-long spacer termed 2O(10), displays high affinity for the cytokine and inhibits IFNgamma-HS binding with an IC(50) of 35-40 nm. Interestingly, this molecule also inhibits the binding of IFNgamma to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNgamma activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNgamma recognition, providing a new strategy to inhibit IFNgamma in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create"tailor-made"sequences based on the HS template to isolate therapeutic activities.     

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor''s response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in α-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.     

Inverted neurons in agyria

Summary An anatomoclinical observation of agyria is reported. The karyotype revealed a partial deletion of the short arm of chromosome 17. The etiology of agyria is reviewed in the light of this chromosomal abnormality. In addition we describe the peculiar pattern of neurons in the cortex: Golgi stain demonstrated many inverted pyramidal cells in the superficial part of the cortical layer. The mechanism of this abnormality is discussed.     

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells

Effects on Ca++ translocation and [Ca++]i were studied in dog thyro?d cell monolayers using both 45Ca++ efflux and the indicator quin-2. Carbamylcholine, a non hydrolysable analog of acetylcholine, through muscarinic receptors, and to a lesser extent TRH and PGF2 alpha increased both these parameters. [Ca++]i increased by 171, 100 and 75% respectively over a basal level of 66 +/- 17 nM (mean +/- SD). The response to carbamylcholine was biphasic. A transient increase in [Ca++]i was followed by a more sustained phase where the [Ca++]i was slightly higher than the basal level. Only the first phase was insensitive to extracellular Ca++ depletion. This phase is probably due to a release of Ca++ from an intracellular store. NaF also induced a sustained rise in [Ca++]i dependent on extracellular Ca++ and affected 45Ca++ efflux. Our data provide direct evidence of an implication of intracellular Ca++ in the response of dog thyro?d cells to all these agents.     

Atomistic mechanisms of huntingtin N‐terminal fragment insertion on a phospholipid bilayer revealed by molecular dynamics simulations

The huntingtin protein is characterized by a segment of consecutive glutamines (Q_N) that is responsible for its fibrillation. As with other amyloid proteins, misfolding of huntingtin is related to Huntington's disease through pathways that can involve interactions with phospholipid membranes. Experimental results suggest that the N‐terminal 17‐amino‐acid sequence (htt^NT) positioned just before the Q_N region is important for the binding of huntingtin to membranes. Through all‐atom explicit solvent molecular dynamics simulations, we unveil the structure and dynamics of the htt^NTQ_N fragment on a phospholipid membrane at the atomic level. We observe that the insertion dynamics of this peptide can be described by four main steps—approach, reorganization, anchoring, and insertion—that are very diverse at the atomic level. On the membrane, the htt^NT peptide forms a stable α‐helix essentially parallel to the membrane with its nonpolar side‐chains—mainly Leu‐4, Leu‐7, Phe‐11 and Leu‐14—positioned in the hydrophobic core of the membrane. Salt‐bridges involving Glu‐5, Glu‐12, Lys‐6, and Lys‐15, as well as hydrogen bonds involving Thr‐3 and Ser‐13 with the phospholipids also stabilize the structure and orientation of the htt^NT peptide. These observations do not significantly change upon adding the Q_N region whose role is rather to provide, through its hydrogen bonds with the phospholipids' head group, a stable scaffold facilitating the partitioning of the htt^NT region in the membrane. Moreover, by staying accessible to the solvent, the amyloidogenic Q_N region could also play a key role for the oligomerization of htt^NTQ_N on phospholipid membranes. Proteins 2014; 82:1409–1427. © 2014 Wiley Periodicals, Inc.     

Seasonal Variation of Antifouling Activities of Marine Algae from the Brittany Coast (France)

The antifouling activity of extracts (aqueous, ethanol, and dichloromethane) of 9 marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae has been investigated in relation to season in bimonthly samples from the Bay of Concarneau (France). Of the extracts tested, 48.2% were active against at least one of the fouling organisms, and of these extracts, 31.2% were seasonally active with a peak of activity in summer corresponding to maximal values for water temperature, light intensity, and fouling pressure, and 17% were active throughout the year. This seasonal activity may be adaptive as it coincides with maximal fouling pressure in the Bay of Concarneau. Dichloromethane extracts of Rhodophyceae were the most active in the antifouling assays.