Genome-scale gene/reaction essentiality and synthetic lethality analysis

Synthetic lethals are to pairs of non-essential genes whose simultaneous deletion prohibits growth. One can extend the concept of synthetic lethality by considering gene groups of increasing size where only the simultaneous elimination of all genes is lethal, whereas individual gene deletions are not. We developed optimization-based procedures for the exhaustive and targeted enumeration of multi-gene (and by extension multi-reaction) lethals for genome-scale metabolic models. Specifically, these approaches are applied to iAF1260, the latest model of Escherichia coli, leading to the complete identification of all double and triple gene and reaction synthetic lethals as well as the targeted identification of quadruples and some higher-order ones. Graph representations of these synthetic lethals reveal a variety of motifs ranging from hub-like to highly connected subgraphs providing a birds-eye view of the avenues available for redirecting metabolism and uncovering complex patterns of gene utilization and interdependence. The procedure also enables the use of falsely predicted synthetic lethals for metabolic model curation. By analyzing the functional classifications of the genes involved in synthetic lethals, we reveal surprising connections within and across clusters of orthologous group functional classifications

Inherent Signals in Sequencing-Based Chromatin-ImmunoPrecipitation Control Libraries

The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.First, we analyzed the genome-wide profiles of various sequencing-based libraries at a low resolution of 1 Mbp, and compared them with each other as well as against aCGH data. We found that copy number plays a major influence in both ChIP-enriched as well as control libraries. Following that, we inspected the repeat regions to assess the extent of mapping bias. Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks. For instance, we discovered that gene boundaries were surprisingly enriched with sequenced tags. Further, profiles between different cell types were noticeably distinct although the cell types were somewhat related and similar.We found that control libraries bear traces of systematic biases. The biases can be attributed to genomic copy number, inherent sequencing bias, plausible mapping ambiguity, and cell-type specific chromatin structure. Our results suggest careful analysis of control libraries can reveal promising biological insights

Sexual conflict maintains variation at an insecticide resistance locus

Background: The maintenance of genetic variation through sexually antagonistic selection is controversial, partly because specific sexually-antagonistic alleles have not been identified. The Drosophila DDT resistance allele (DDT-R) is an exception. This allele increases female fitness, but simultaneously decreases male fitness, and it has been suggested that this sexual antagonism could explain why polymorphism was maintained at the locus prior to DDT use. We tested this possibility using a genetic model and then used evolving fly populations to test model predictions. Results: Theory predicted that sexual antagonism is able to maintain genetic variation at this locus, hence explaining why DDT-R did not fix prior to DDT use despite increasing female fitness, and experimentally evolving fly populations verified theoretical predictions. Conclusions: This demonstrates that sexually antagonistic selection can maintain genetic variation and explains the DDT-R frequencies observed in nature

Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

Background: During a normal cell cycle, the transition from G 2 phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes. Methodology/Principal Findings: It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest. Conclusions/Significance: These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associate

QTL Mapping of Combining Ability and Heterosis of Agronomic Traits in Rice Backcross Recombinant Inbred Lines and Hybrid Crosses

BACKGROUND: Combining ability effects are very effective genetic parameters in deciding the next phase of breeding programs. Although some breeding strategies on the basis of evaluating combining ability have been utilized extensively in hybrid breeding, little is known about the genetic basis of combining ability. Combining ability is a complex trait that is controlled by polygenes. With the advent and development of molecular markers, it is feasible to evaluate the genetic bases of combining ability and heterosis of elite rice hybrids through QTL analysis. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we first developed a QTL-mapping method for dissecting combining ability and heterosis of agronomic traits. With three testcross populations and a BCRIL population in rice, biometric and QTL analyses were conducted for ten agronomic traits. The significance of general combining ability and special combining ability for most of the traits indicated the importance of both additive and non-additive effects on expression levels. A large number of additive effect QTLs associated with performance per se of BCRIL and general combining ability, and dominant effect QTLs associated with special combining ability and heterosis were identified for the ten traits. CONCLUSIONS/SIGNIFICANCE: The combining ability of agronomic traits could be analyzed by the QTL mapping method. The characteristics revealed by the QTLs for combining ability of agronomic traits were similar with those by multitudinous QTLs for agronomic traits with performance per se of BCRIL. Several QTLs (1-6 in this study) were identified for each trait for combining ability. It demonstrated that some of the QTLs were pleiotropic or linked tightly with each other. The identification of QTLs responsible for combining ability and heterosis in the present study provides valuable information for dissecting genetic basis of combining ability

Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid

Background: The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance. Methods: BAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene. Results: Human 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR. Conclusion: The Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.Supported by NIH grant R01 AI054703 from the National Institute of Allergy and Infectious Diseases

The sex locus is tightly linked to factors conferring sex-specific lethal effects in the mosquito Aedes aegypti

In many taxa, sex chromosomes are heteromorphic and largely non-recombining. Evolutionary models predict that spread of recombination suppression on the Y chromosome is fueled by the accumulation of sexually antagonistic alleles in close linkage to the sex determination region. However, empirical evidence for the existence of sexually antagonistic alleles is scarce. In the mosquito Aedes aegypti, the sex-determining chromosomes are homomorphic. The region of suppressed recombination, which surrounds the male-specific sex-determining gene, remains very small, despite ancient origin of the sex chromosomes in the Aedes lineage. We conducted a genetic analysis of the A. aegypti chromosome region tightly linked to the sex locus. We used a strain with an enhanced green fluorescent protein (EGFP)-tagged transgene inserted near the male-determining gene to monitor crossing-over events close to the boundary of the sex-determining region (SDR), and to trace the inheritance pattern of the transgene in relation to sex. In a series of crossing experiments involving individuals with a recombinant sex chromosome we found developmental abnormalities leading to 1:2 sex biases, caused by lethality of half of the male or female progeny. Our results suggest that various factors causing sex-specific lethal effects are clustered within the neighborhood of the SDR, which in the affected sex are likely lost or gained through recombination, leading to death. These may include genes that are recessive lethal, vital for development and/or sexually antagonistic. The sex chromosome fragment in question represents a fascinating test case for the analysis of processes that shape stable boundaries of a non-recombining region

The Impact of CpG Island on Defining Transcriptional Activation of the Mouse L1 Retrotransposable Elements

BACKGROUND: L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500-3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active T(F) family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome. CONCLUSIONS: A comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements

Health Related Quality of Life among Patients with Tuberculosis and HIV in Thailand

INTRODUCTION: Health utilities of tuberculosis (TB) patients may be diminished by side effects from medication, prolonged treatment duration, physical effects of the disease itself, and social stigma attached to the disease. METHODS: We collected health utility data from Thai patients who were on TB treatment or had been successfully treated for TB for the purpose of economic modeling. Structured questionnaire and EuroQol (EQ-5D) and EuroQol visual analog scale (EQ-VAS) instruments were used as data collection tools. We compared utility of patients with two co-morbidities calculated using multiplicative model (U(CAL)) with the direct measures and fitted Tobit regression models to examine factors predictive of health utility and to assess difference in health utilities of patients in various medical conditions. RESULTS: Of 222 patients analyzed, 138 (62%) were male; median age at enrollment was 40 years (interquartile range [IQR], 35-47). Median monthly household income was 6,000 Baht (187 US$; IQR, 4,000-15,000 Baht [125-469 US$]). Concordance correlation coefficient between utilities measured using EQ-5D and EQ-VAS (U(EQ-5D) and U(VAS), respectively) was 0.6. U(CAL) for HIV-infected TB patients was statistically different from the measured U(EQ-5D) (p-value<0.01) and U(VAS) (p-value<0.01). In tobit regression analysis, factors independently predictive of U(EQ-5D) included age and monthly household income. Patients aged ≥40 years old rated U(EQ-5D) significantly lower than younger persons. Higher U(EQ-5D) was significantly associated with higher monthly household income in a dose response fashion. The median U(EQ-5D) was highest among patients who had been successfully treated for TB and lowest among multi-drug resistant TB (MDR-TB) patients who were on treatment. CONCLUSIONS: U(CAL) of patients with two co-morbidities overestimated the measured utilities, warranting further research of how best to estimate utilities of patients with such conditions. TB and MDR-TB treatments impacted on patients' self perceived health status. This effect diminished after successful treatment

Effects of NFKB1 and NFKBIA Gene Polymorphisms on Susceptibility to Environmental Factors and the Clinicopathologic Development of Oral Cancer

encoding IkappaBalpha (IκBα) with both the susceptibility to develop OSCC and the clinicopathological characteristics of the tumors.<.05), compared with those patients CC homozygotes. 519 might be a predictive factor for the distal metastasis of OSCC in Taiwanese