A genome-wide screening in Saccharomyces cerevisiae for genes that confer resistance to the anticancer agent cisplatin

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.     

Upregulation of hepatic glucose 6-phosphatase gene expression in rats treated with an inhibitor of glucose-6-phosphate translocase

The multicomponent hepatic glucose 6-phosphatase (Glc-6-Pase) system catalyzes the terminal step of hepatic glucose production and plays a key role in the regulation of blood glucose. We used the chlorogenic acid derivative S 3483, a reversible inhibitor of the glucose-6-phosphate (Glc-6-P) translocase component, to demonstrate for the first time upregulation of Glc-6-Pase expression in rat liver in vivo after inhibition of Glc-6-P translocase. In accordance with its mode of action, S 3483-treatment of overnight-fasted rats induced hypoglycemia and increased blood lactate, hepatic Glc-6-P, and glycogen. The metabolic changes were accompanied by rapid and marked increases in Glc-6-Pase mRNA (above 35-fold), protein (about 2-fold), and enzymatic activity (about 2-fold). Maximal mRNA levels were reached after 4 h of treatment. Glycemia, blood lactate, and Glc-6-Pase mRNA levels returned to control values, whereas Glc-6-P and glycogen levels decreased but were still elevated 2 h after S 3483 withdrawal. The capacity for Glc-6-P influx was only marginally increased after 8.5 h of treatment. Prevention of hypoglycemia by euglycemic clamp did not abolish the increase in Glc-6-Pase mRNA induced by S 3483 treatment. A similar pattern of hypoglycemia and possibly of associated counterregulatory responses elicited by treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinic acid could account for only a 2-fold induction of Glc-6-Pase mRNA. These findings suggest that the significant upregulation of Glc-6-Pase gene expression observed after treatment of rats in vivo with an inhibitor of Glc-6-P translocase is caused predominantly either by S 3483 per se or by the compound-induced changes of intracellular carbohydrate metabolism.     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

Phylogenomic Analysis Reveals Deep Divergence and Recombination in an Economically Important Grapevine Virus

The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.     

Socio-Economic Instability and the Scaling of Energy Use with Population Size

The size of the human population is relevant to the development of a sustainable world, yet the forces setting growth or declines in the human population are poorly understood. Generally, population growth rates depend on whether new individuals compete for the same energy (leading to Malthusian or density-dependent growth) or help to generate new energy (leading to exponential and super-exponential growth). It has been hypothesized that exponential and super-exponential growth in humans has resulted from carrying capacity, which is in part determined by energy availability, keeping pace with or exceeding the rate of population growth. We evaluated the relationship between energy use and population size for countries with long records of both and the world as a whole to assess whether energy yields are consistent with the idea of an increasing carrying capacity. We find that on average energy use has indeed kept pace with population size over long time periods. We also show, however, that the energy-population scaling exponent plummets during, and its temporal variability increases preceding, periods of social, political, technological, and environmental change. We suggest that efforts to increase the reliability of future energy yields may be essential for stabilizing both population growth and the global socio-economic system.     

Roles of the main olfactory and vomeronasal systems in the detection of androstenone in inbred strains of mice

We investigated the role of the main olfactory and accessory olfactory systems (MOS and AOS respectively) in the detection of androstenone. We used the following experimental approaches: behavioral, surgical removal of the vomeronasal organ (VNX) followed by histochemical verification and Fos immunohistochemistry. Using a Y-maze paradigm we estimated sensitivity of NZB/B1NJ and CBA/J mice to androstenone. CBA mice were 2,000-fold more sensitive to androstenone than NZB mice. VNX caused a 4- to 16-fold decre...     

DNA barcodes reveal species-specific mercury levels in tuna sushi that pose a health risk to consumers

Excessive ingestion of mercury—a health hazard associated with consuming predatory fishes—damages neurological, sensory-motor and cardiovascular functioning. The mercury levels found in Bigeye Tuna (Thunnus obesus) and bluefin tuna species (Thunnus maccoyii, Thunnus orientalis, and Thunnus thynnus), exceed or approach levels permissible by Canada, the European Union, Japan, the US, and the World Health Organization. We used DNA barcodes to identify tuna sushi samples analysed for mercury and demonstrate that the ability to identify cryptic samples in the market place allows regulatory agencies to more accurately measure the risk faced by fish consumers and enact policies that better safeguard their health.     

Assessing and reporting heterogeneity in treatment effects in clinical trials: a proposal

Background

This paper presents the study protocol for a pragmatic randomised controlled trial to evaluate the impact of a school based program developed to prevent teenage pregnancy. The program includes students taking care of an Infant Simulator; despite growing popularity and an increasing global presence of such programs, there is no published evidence of their long-term impact. The aim of this trial is to evaluate the Virtual Infant Parenting (VIP) program by investigating pre-conceptual health and risk behaviours, teen pregnancy and the resultant birth outcomes, early child health and maternal health.

Methods and Design

Fifty-seven schools (86% of 66 eligible secondary schools) in Perth, Australia were recruited to the clustered (by school) randomised trial, with even randomisation to the intervention and control arms. Between 2003 and 2006, the VIP program was administered to 1,267 participants in the intervention schools, while 1,567 participants in the non-intervention schools received standard curriculum. Participants were all female and aged between 13-15 years upon recruitment. Pre and post-intervention questionnaires measured short-term impact and participants are now being followed through their teenage years via data linkage to hospital medical records, abortion clinics and education records. Participants who have a live birth are interviewed by face-to-face interview. Kaplan-Meier survival analysis and proportional hazards regression will test for differences in pregnancy, birth and abortion rates during the teenage years between the study arms.

Discussion

This protocol paper provides a detailed overview of the trial design as well as initial results in the form of participant flow. The authors describe the intervention and its delivery within the natural school setting and discuss the practical issues in the conduct of the trial, including recruitment. The trial is pragmatic and will directly inform those who provide Infant Simulator based programs in school settings.

Trial registration

ISRCTN24952438     

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and ‘Dulce’ (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.