A simple but highly effective approach to evaluate the prognostic performance of gene expression signatures

Background

Highly parallel analysis of gene expression has recently been used to identify gene sets or ‘signatures’ to improve patient diagnosis and risk stratification. Once a signature is generated, traditional statistical testing is used to evaluate its prognostic performance. However, due to the dimensionality of microarrays, this can lead to false interpretation of these signatures.

Principal Findings

A method was developed to test batches of a user-specified number of randomly chosen signatures in patient microarray datasets. The percentage of random generated signatures yielding prognostic value was assessed using ROC analysis by calculating the area under the curve (AUC) in six public available cancer patient microarray datasets. We found that a signature consisting of randomly selected genes has an average 10% chance of reaching significance when assessed in a single dataset, but can range from 1% to ∼40% depending on the dataset in question. Increasing the number of validation datasets markedly reduces this number.

Conclusions

We have shown that the use of an arbitrary cut-off value for evaluation of signature significance is not suitable for this type of research, but should be defined for each dataset separately. Our method can be used to establish and evaluate signature performance of any derived gene signature in a dataset by comparing its performance to thousands of randomly generated signatures. It will be of most interest for cases where few data are available and testing in multiple datasets is limited.     

Intra-Clutch Ratio of Yolk Progesterone Level Changes with Laying Date in Rockhopper Penguins: A Strategy to Influence Brood Reduction?

Hatching asynchrony in avian species generally leads to a size hierarchy among siblings, favouring the first-hatched chicks. Maternally deposited hormones affect the embryo and chick's physiology and behaviour. It has been observed that progesterone, a hormone present at higher levels than other steroid hormones in egg yolks, is negatively related to body mass in embryos, chicks and adults. A differential within-clutch progesterone deposition could therefore be linked to the size hierarchy between siblings and to the resulting brood reduction. We tested whether yolk progesterone levels differed between eggs according to future parental ability to feed the entire clutch in wild rockhopper penguins Eudyptes chrysocome. This species presents a unique reversed egg-size dimorphism and hatching asynchrony, with the larger second-laid egg (B-egg) hatching before the smaller first-laid egg (A-egg). Yolk progesterone levels increased only slightly with female body mass at laying. However, intra-clutch ratios were not related to female body mass. On the other hand, yolk progesterone levels increased significantly with the date of laying onset for A-eggs while they decreased for B-eggs. Early clutches therefore had proportionally more progesterone in the B-egg compared to the A-egg while late clutches had proportionally less progesterone in the B-egg. We propose that females could strategically regulate yolk progesterone deposition within clutches according to the expected food availability during chick growth, an adaptive strategy to adjust brood reduction to conditions. We also discuss these results, relating to yolk progesterone, in the broader context of other yolk steroids.     

The influence of day-length on imaginal diapause in the Red Locust, Nomadacris septemfasciata (Serv.)

The main permanent habitats of the Red Locust (Nomadacris septemfasciata (Serv.)) in tropical Africa lie between latitudes 7° and 9° S and between 10° and 16° N. Breeding is confined to the rainy season, which occurs during the longest days of the year in these areas. Both north and south of the equator, the adults emerge as day-length is diminishing and undergo a prolonged imaginal diapause until the next rainy season.When reared in the laboratory in London, adults which emerge in spring or early summer become sexually mature within two months; those which emerge in late summer and autumn, although kept at the same temperature and humidity, enter diapause and do not copulate or oviposit until the following spring. By controlling the length of the daily photoperiod, it has been shown that the seasonal differences in day-length are responsible for the onset of diapause. Constant short photoperiods usually induce a rather shorter diapause than photoperiods diminishing so as to simulate autumn day-length at this latitude (51.5° N). The critical constant photoperiod for the establishment of diapause lies between 12 and 13 hours which corresponds approximately to maximum and minimum day-lengths at 7° N or S. It is therefore possible that day-length is responsible for the establishment of the natural diapause.

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Resume On a déjà constaté que, quand on élève le criquet rouge, Nomadacris septemfasciata (Serv.), au laboratoire à Londres, les adultes, malgré le fait qu'on les retient dans une ambiance très constante, montrent de grandes différences saisonnières dans la durie du développement qui conduit à la maturité sexuelle. Les adultes qui émergent à partir du mois d'avril jusque'au mois de juillet deviennent matures sexuellement sans diapause en 11/2 à 2 mois; tandis que ceux qui émergent au début de l'automne, à partir de la mi-août approximativement jusqu'à la fin de septembre, ont une diapause prolongée de 7 à 8 mois; et ceux qui émergent encore plus tard dans l'année, du mois d'octobre jusqu'à mi-décembre, ont une diapause plus courte de 41/2 à 51/2 mois. Les périodes de maturation des adultes qui émergent à partir de janvier et plus tard, diminuent progressivement, en se fondant dans les groupes des non-diapause, qui commencent à émerger au début d'avril. Les expériences exposées dans cette thèse indiquent que les différences saisonnières de la longueur des jours, sont responsables des temps de maturation.Certains groupes de criquets, émergeant comme adultes vers la fin de l'été ou en automne, ont été soumis à des journées de longueur artificielle qui correspondait à la durée des jours de printems qui n'exerce son influence que sur la vie adulte, suffit à produire ce dans les délais semblables à ceux des groupes qui émergent au printemps. La longueur des jours de printemps qui n'excerce son influence que sur la vie adulte, suffit à produire ce résultat.Quelques larves étaient élevées simultanément dans les conditions d'une durée artificielle des jours du printemps, d'une durée artificielle des jours d'automne, et d'une lumière naturelle des jours d'été à Londres. Les adultes qui avaient émergé au mois de juin étaient ou maintenus dans le même régime de longueur de journée que les larves, ou transférés dans une des autres conditions. Tous les temps de maturation étaient appropriés aux saisons qui correspondaient au régime de durée du jour auquel les adultes étaient retenus, et il n'y avait nulle évidence qu'ils étaient influencés par la durée des jours pendant leur vie larvaire.Les groupes diapause, quand ils étaient soumis à la lumière normale du laboratoire, sont devenus matures, au printemps, au moment où les jours s'allongeaient. Pourtant la maturation n'est pas due à la stimulation causée par un accroissement de la longueur du jour, car on à observé qu'elle arrive en même temps si les criquets ne la subissent pas. Ceci est vrai également à l'égard des deux groupes, celui de longue diapause (qui émerge au début de l'automne) et celui de courte diapause (qui émerge vers la fin de l'automne); il s'ensuit que la diapause plus courte du dernier groupe n'est pas attribuable, comme il semblait probable, au fait qu'on l'avait exposé à la longueur des jours du printemps plus tôt dans la vie adulte. On a trouvé que ces groupes de la dernière partie de l'automne étaient devenus matures dans le même temps s'ils étaient retenus aux courtes photo-périodes constantes de 8 ou 11 heures, ou à la durée changeante des jours d'hiver. On a conclu qu'ils répondent comme à la durée de constants jours courts.Les groupes intermédaires (qui émergent à partir de janvier jusqu'a mars) d'autre part, sont exposés aux jours plus longs du printemps et de l'été encore plus tôt dans la vie adulte, et dans leur cas la diapause est empêchée, ou au moins abrégée, par les jours plus longs. Quand ces groupes sont retenus aux constantes photo-périodes courtes, ils deviennent matures plus lentement que s'ils étaient maintenus à la lumière du jour, et ils ont une diapause de la même longueur que les groupes de courte-diapause.On a conclu de ces résultats que, quand les groupes sont retenus dans conditions de jours-courts, les processus de maturation (manifestés par un accroissement du poids du corps, et par le développement de l'ovaire) sont repris à un certain stade indépendamment de la longueur du jour et sous l'action de stimuli internes. L'exposition aux conditions de jours longs n'est effective que pour accélérer la maturation si elle a lieu avant que ce stade soit atteint.L'observation que les adultes émergeant au début de l'automne ont une diapause plus longue que ceux qui émergent vers la fin de l'automne, reste sans explication. Ceux-là sont exposés à la durée de jours qui décroissent rapidement pendant la vie larvaire et du commencement de la vie adulte, et il semble probable que le changement propre des jours longs aux jours courts puisse amener une diapause plus longue que ne le feraient des jours courts dès le début. A présent, on étudie les effets de la variation de durée des jours.Les adultes retenus dans l'obscurité constante sont devenus matures sans diapause.La grande fluctuation de la longueur des jours, caractéristique de la latitude de Londres, n'est pas nécessaire pour induire les deux types de maturation diapause ou non-diapause. Les plus grandes différences de réponse aux photo-périodes constantes ont eu lieu entre les moyennes correspondant à peu près avec les minimum et maximum de longueurs de jour (y compris les périodes de crépuscule) dans les habitats tropicaux du criquet.Il est discuté ici de la probabilité que le cours de la diapause dans l'habitat tropical naturel soit influencé par la faible fluctuation des longueurs de jour de cet habitat.

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The author is Mrs O. W. Richards     

Nuclear reprogramming and adult stem cell potential

Cell-based therapy may represent a new strategy to treat a vast array of clinical disorders including neurodegenerative diseases. Recent observations indicate that adult somatic stem cells have the capacity to contribute to the regeneration of different tissues, suggesting that differentiative restrictions are not completely irreversible and can be reprogrammed. Cell fusion might account for some changed phenotype of adult cells but it seems to be biologically irrelevant for its extreme rarity. Other experimental evidences are compatible with the hypothesis of wide multipotency of well-defined stem cell populations, but also with transdifferentiation and/or dedifferentiation. Further studies on nuclear reprogramming mechanisms are necessary to fulfil the promise for developing autologous cellular therapies.     

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.     

Solid-phase microextraction and chiral HPLC analysis of ibuprofen in urine

A simple and rapid solid-phase microextraction method was developed for the enantioselective analysis of ibuprofen in urine. The sampling was made with a polydimethylsiloxane-divinylbenzene coated fiber immersed in the liquid sample. After desorptioning from the fiber, ibuprofen enantiomers were analyzed by HPLC using a Chiralpak AD-RH column and UV detection. The mobile phase was made of methanol-pH 3.0 phosphoric acid solution (75:25, v/v), at a flow rate of 0.45 mL/min. The mean recoveries of SPME were 19.8 and 19.1% for (-)-R-ibuprofen and (+)-(S)-ibuprofen, respectively. The method was linear at the range of 0.25-25 microg/mL. Within-day and between-day assay precision and accuracy were below 15% for both ibuprofen enantiomers at concentrations of 0.75, 7.5 and 20 microg/mL. The method was tested with urine quality control samples and human urine fractions after administration of 200 mg rac-ibuprofen.     

Subplasmalemmal mitochondria modulate the activity of plasma membrane Ca2+-ATPases

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.     

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 microM verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent (109)Cd(2+) uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metal-extrusion abilities.     

Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels

Consumption of plant sterols and treatment with ezetimibe both reduce cholesterol absorption in the intestine. However, the mechanism of action differs between the two treatments, and the consequences of combination treatment are unknown. Therefore, we performed a double-blind, placebo-controlled, crossover study for the plant sterol component with open-label ezetimibe treatment. Forty mildly hypercholesterolemic subjects were randomized to the following treatments for 4 weeks each: 10 mg/day ezetimibe combined with 25 g/day control spread; 10 mg/day ezetimibe combined with 25 g/day spread containing 2.0 g of plant sterols; 25 g/day spread containing 2.0 g of plant sterols; and placebo treatment consisting of 25 g/day control spread. Combination treatment of plant sterols and ezetimibe reduced low density lipoprotein cholesterol (LDL-C) by 1.06 mmol/l (25.2%; P < 0.001) compared with 0.23 mmol/l (4.7%; P = 0.006) with plant sterols and 0.94 mmol/l (22.2%; P < 0.001) with ezetimibe monotherapy. LDL-C reduction conferred by the combination treatment did not differ significantly from ezetimibe monotherapy (-0.12 mmol/l or -3.5%; P = 0.13). Additionally, the plasma lathosterol-to-cholesterol ratio increased with all treatments. Sitosterol and campesterol ratios increased after plant sterol treatment and decreased upon ezetimibe and combination therapy. Our results indicate that the combination of plant sterols and ezetimibe has no therapeutic benefit over ezetimibe monotherapy in subjects with mild hypercholesterolemia.