Influence of trace erythromycin and eryhthromycin-H2O on carbon and nutrients removal and on resistance selection in sequencing batch reactors (SBRs).

Three sequencing batch reactors (SBRs) were operated in parallel to study the effects of trace erythromycin (ERY) and ERY-H2O on the treatment of a synthetic wastewater. Through monitoring (1) daily effluents and (2) concentrations of nitrogen (N) and phosphorous (P) in certain batch cycles of the three reactors operated from transient to steady states, the removal of carbon, N, and P was affected negligibly by ERY (100 microg/L) or ERY-H2O (50 microg/L) when compared with the control reactor. However, through analyzing microbial communities of the three steady state SBRs on high-density microarrays (Phylo-Chip), ERY, and ERY-H2O had pronounced effects on the community composition of bacteria related to N and P removal, leading to diversity loss and abundance change. The above observations indicated that resistant bacteria were selected upon exposure to ERY or ERY-H2O. Shortterm batch experiments further proved the resistance and demonstrated that ammonium oxidation (56-95%) was inhibited more significantly than nitrite oxidation (18-61%) in the presence of ERY (100, 400, or 800 microg/L). Therefore, the presence of ERY or ERY-H2O (at microg/L levels) shifted the microbial community and selected resistant bacteria, which may account for the negligible influence of the antibiotic ERY or its derivative ERY-H2O (at microg/L levels) on carbon, N, and P removal in the SBRs

Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea

<p>Abstract</p> <p>Background</p> <p>Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of <it>L. hongkongensis</it>. In this study, a multilocus sequence typing (MLST) system was developed for <it>L. hongkongensis</it>. The system was used to characterize 146 <it>L. hongkongensis </it>isolates, including 39 from humans and 107 from fish.</p> <p>Results</p> <p>Fragments (362 to 504 bp) of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (<it>ilvC</it>) to 45 (<it>thiC</it>)]. All seven genes showed very low <it>d</it>_<it>n</it>/<it>d</it>_{<it>s </it>}ratios of < 0.04, indicating that no strong positive selective pressure is present. A total of 97 different sequence types (STs) were assigned to the 146 isolates, with 80 STs identified only once. The overall discriminatory power was 0.9861. eBURST grouped the isolates into 12 lineages, with six groups containing only isolates from fish and three groups only isolates from humans. Standardized index of association (<it>I</it>^<it>S</it>_<it>A</it>) measurement showed significant linkage disequilibrium in isolates from both humans and fish. The <it>I</it>^<it>S</it>_{<it>A </it>}for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (<it>acnB</it>) was detected in the split graphs. The P-value (P = 0) of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the <it>rho, acnB </it>and <it>thiC </it>loci, but the P-value (P = 1) of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial role in the evolution of <it>L. hongkongensis</it>. A website for <it>L. hongkongensis </it>MLST was set up and can be accessed at <url>http://mlstdb.hku.hk:14206/MLST_index.html</url>.</p> <p>Conclusion</p> <p>A highly reproducible and discriminative MLST system was developed for <it>L. hongkongensis</it>.</p

Refinement of Light-Responsive Transcript Lists Using Rice Oligonucleotide Arrays: Evaluation of Gene-Redundancy

Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org). The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics

Identification and Functional Analysis of Light-Responsive Unique Genes and Gene Family Members in Rice

Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families

Towards Establishment of a Rice Stress Response Interactome

Rice (Oryza sativa) is a staple food for more than half the world and a model for studies of monocotyledonous species, which include cereal crops and candidate bioenergy grasses. A major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%–60% yield losses globally each year. To elucidate stress response signaling networks, we constructed an interactome of 100 proteins by yeast two-hybrid (Y2H) assays around key regulators of the rice biotic and abiotic stress responses. We validated the interactome using protein–protein interaction (PPI) assays, co-expression of transcripts, and phenotypic analyses. Using this interactome-guided prediction and phenotype validation, we identified ten novel regulators of stress tolerance, including two from protein classes not previously known to function in stress responses. Several lines of evidence support cross-talk between biotic and abiotic stress responses. The combination of focused interactome and systems analyses described here represents significant progress toward elucidating the molecular basis of traits of agronomic importance

The Efficacy of Generating Three Independent Anti-HIV-1 siRNAs from a Single U6 RNA Pol III-Expressed Long Hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs

Association between physical activity and metabolic syndrome: a cross sectional survey in adolescents in Ho Chi Minh City, Vietnam

<p>Abstract</p> <p>Background</p> <p>The emerging epidemic of overweight/obesity in adolescents in Ho Chi Minh City, Vietnam underlines the importance of studying the metabolic syndrome in Vietnamese adolescents who are becoming progressively more inactive. No study in Vietnam has examined the association of metabolic syndrome with moderate to vigorous physical activity (PA) levels among adolescents. We aimed to examine this association in a sample of urban adolescents from Ho Chi Minh City.</p> <p>Methods</p> <p>A cross-sectional assessment was conducted in 2007 on a representative sample of 693 high-school students from urban districts in Ho Chi Minh City. Metabolic syndrome was defined according to the International Diabetes Federation criteria and physical activity was measured with Actigraph accelerometers. The association between physical activity and metabolic syndrome was assessed by using multiple logistic regression models.</p> <p>Results</p> <p>Overall 4.6% of the adolescents and 11.8% of the overweight/obese adolescents had metabolic syndrome. Elevated BP was the most common individual component of the metabolic syndrome (21.5%), followed by hypertriglyceridemia (11.1%). After adjusting for other study factors, the odds of metabolic syndrome among youth in the lowest physical activity group (<43 minutes of physical activity/day) were five times higher than those in the highest physical activity group (>103 minutes/day) (AOR = 5.3, 95% CI: 1.5, 19.1). Metabolic syndrome was also positively associated with socioeconomic status (AOR = 9.4, 95% CI: 2.1, 42.4).</p> <p>Conclusions</p> <p>A more physically active lifestyle appears to be associated with a lower odds of metabolic syndrome in Vietnamese adolescents. Socio-economic status should be taken into account when planning interventions to prevent adolescent metabolic syndrome.</p

Nkx2.7 and Nkx2.5 Function Redundantly and Are Required for Cardiac Morphogenesis of Zebrafish Embryos

Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear.Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO.Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage

OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis