Exon-primed intron-crossing (EPIC) PCR markers of Helicoverpa armigera (Lepidoptera: Noctuidae)

Applying microsatellite DNA markers in population genetic studies of the pest moth Helicoverpa armigera is subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers for H. armigera based on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele 'dropouts'. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species, H. zea, H. assulta and H. punctigera.     

Enhanced Survival of Melanopsin-expressing Retinal Ganglion Cells After Injury is Associated with the PI3 K/Akt Pathway

In the present study, we studied the factors that contribute to the injury-resistant property of melanopsin-expressing retinal ganglion cells (mRGCs). Since phosphatidylinositol-3 kinase (PI3 K)/Akt signaling pathway is one of the well-known pathways for neuronal cell survival, we investigated the survival of mRGCs by applying the PI3 K/Akt specific inhibitors after injury. Two injury models, unilateral optic nerve transection and ocular hypertension, were adopted using Sprague-Dawley rats. Inhibitors of PI3 K/Akt were injected intravitreally following injuries to inhibit the PI3 K/Akt signaling pathway. Retinas were dissected after designated survival time, immunohistochemistry was carried out to visualize the mRGCs using melanopsin antibody and the number of mRGCs was counted. Co-expression of melanopsin and phospho-Akt (pAkt) was also examined. Compared to the survival of non-melanopsin-expressing RGCs, mRGCs showed a marked resistance to injury and co-expressed pAkt. Application of PI3 K/Akt inhibitors decreased the survival of mRGCs after injury. Our previous study has shown that mRGC are less susceptible to injury following the induction of ocular hypertension. In this study, we report that mRGCs were injury-resistant to a more severe type of injury, the optic nerve transection. More importantly, the PI3 K/Akt pathway was found to play a role in maintaining the survival of mRGCs after injury.     

Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats

The extent of in vivo oxidative damage has been known to be cumulative over the period of the life of mammals. Our hypothesis is that there should be a positive correlation between the levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in major rat tissues. We also investigated whether increased level of oxidative stress causes a decrease in the mitochondrial membrane potential of peripheral lymphocytes of old rats using the MitoTracker Red fluorochrome. Our results show positive correlations between 8OHdG and 8-iso-PGF(2alpha) for liver, brain and kidney measured by HPLC-UV-ECD (electrochemical detector) and EIA methods, respectively. However, heart tissues show a negative correlation. The mitochondrial membrane potential of old rat lymphocytes records significant decrease compared with the young lymphocytes. Based on our results, we conclude that in ageing studies, specific tissues need to be examined in order to measure the localised DNA damage and lipid peroxidation as different tissues display different extent of oxidative damage. We believe this approach of using combined markers is useful to verify the true efficacy of health intervention studies in animals and humans. In addition, the isoprostane assay can be further developed looking at lipid peroxidation as a potential marker in ageing studies.     

Antimicrobial activity of extracts of the lichen Xanthoparmelia pokornyi and its gyrophoric and stenosporic acid constituents

The antimicrobial activity of the diethyl ether, acetone, chloroform, petroleum ether, and ethanol extracts of the lichen Xanthoparmelia pokornyi and its gyrophoric acid and stenosporic acid constituents has been screened against some foodborne bacteria and fungi. Both the extracts and the acids showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans and Candida glabrata. The extracts were inactive against the tested filamentous fungi. The MIC values of the extracts and the acids for the bacteria have also been determined.     

Effect of dense gas CO2 on the coacervation of elastin

In this study for the first time the effect of high-pressure CO2 on the coacervation of alpha-elastin was investigated using analytical techniques including light spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging and circular dichroism (CD) spectroscopy. The coacervation behavior of alpha-elastin, a protein biopolymer, was determined at temperatures below 40 degrees C and pressures lower than 180 bar. At these conditions elevated pressures did not disrupt the ability of alpha-elastin to coacervate. It was feasible to monitor the presence of amide I, II, and III bands for alpha-elastin at high-pressure CO2 using ATR-FTIR imaging. At a constant temperature the peak absorption was substantially enhanced by increasing the pressure of the system. CD analysis demonstrated the preservation of secondary structure attributes of alpha-elastin exposed to dense gas CO2 at the pressure range investigated in this study. The lower critical solution temperature of alpha-elastin was dramatically decreased from 37 to 16 degrees C when the CO2 pressure increased from 1 to 50 bar, without a significant change after that. Carbon dioxide at high pressures also impeded the reversible coacervation of alpha-elastin solution. These effects were predominantly associated with the lowered pH of the aqueous solution and maybe the interaction between CO2 and hydrophobic domains of alpha-elastin.     

Molecular Characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> Cu/Zn-Superoxide Dismutases Reveals Their Regulation of Shoot Regeneration

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals (O₂^-) to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). In this study we characterized an Arabidopsis thaliana CuZnSOD (CSD1), a close ortholog of a previously identified Brassica juncea CuZnSOD (MSOD1). CSD1 and other two homologs CSD2 and CSD3 were spatially regulated in Arabidopsis, and CSD1 exhibited distinct expression patterns in response to different stress treatments. To investigate the in vivo function of SOD, transgenic Arabidopsis plants, expressing sense and antisense MSOD1 RNAs, were generated and those with altered SOD activity were selected for further characterization. Although SOD transgenic plants exhibited normal phenotypes, the shoot regeneration response in transgenic explants was significantly affected by the modulated SOD activity and the corresponding H₂O₂ levels. Transgenic explants with downregulated SOD activity were poorly regenerative, whereas those with upregulated SOD activity were highly regenerative. These results suggest that shoot regeneration in vitro is regulated by the SOD activity.     

Phosphate removal from the returned liquor of municipal wastewater treatment plant using iron-reducing bacteria

AIM: The application of iron-reducing bacteria (IRB) to phosphate removal from returned liquor (liquid fraction after activated sludge digestion and anaerobic sludge dewatering) of municipal wastewater treatment plant (WWTP) was studied. METHODS AND RESULTS: An enrichment culture and two pure cultures of IRB, Stenotrophomonas maltophilia BK and Brachymonas denitrificans MK identified by 16S rRNA gene sequencing, were produced using returned liquor from a municipal WWTP as carbon and energy source, and iron hydroxide as oxidant. The final concentration of phosphate increased from 70 to 90 mg l(-1) in the control and decreased from 70 to 1 mg l(-1) in the experiment. The mass ratio of removed P to produced Fe(II) was 0.17 g P g(-1) Fe(II). The strain S. maltophilia BK showed the ability to reduce Fe(III) using such xenobiotics as diphenylamine, m-cresol, 2,4-dichlorphenol and p-phenylphenol as sole sources of carbon under anaerobic conditions. CONCLUSIONS: Bacterial reduction of ferric hydroxide enhanced the phosphate removal from the returned liquor. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the facultative anaerobes S. maltophilia BK and B. denitrificans MK to reduce Fe(III) was shown. These micro-organisms can be used for anaerobic removal of phosphate and xenobiotics by bacterial reduction of ferric ions.     

Divide-and-conquer approach for the exemplar breakpoint distance

MOTIVATION: A one-to-one correspondence between the sets of genes in the two genomes being compared is necessary for the notions of breakpoint and reversal distances. To compare genomes where there are paralogous genes, Sankoff formulated the exemplar distance problem as a general version of the genome rearrangement problem. Unfortunately, the problem is NP-hard even for the breakpoint distance. RESULTS: This paper proposes a divide-and-conquer approach for calculating the exemplar breakpoint distance between two genomes with multiple gene families. The combination of our approach and Sankoff's branch-and-bound technique leads to a practical program to answer this question. Tests with both simulated and real datasets show that our program is much more efficient than the existing program that is based only on the branch-and-bound technique. AVAILABILITY: Code for the program is available from the authors.     

Microbial adaptation to biodegradation of tert-butyl alcohol in a sequencing batch reactor

This study demonstrates the utility of the sequencing batch reactor (SBR) to adapt microorganisms towards biological removal of tert-butyl alcohol (TBA). The reactor was inoculated with activated sludge and fed with TBA as the sole carbon source. Start-of-cycle TBA concentrations were initially set at 100 mgL(-1) with a cycle time of 24 h and a volumetric exchange ratio of 50% to maintain a TBA loading rate of not more than 100 mgL(-1)d(-1). Step increases in TBA loading rates up to 600 mgL(-1)d(-1) were achieved by first raising the start-of-cycle TBA concentration to 150 mgL(-1) on day 90 and subsequently by reducing the cycle time from 24 to 12, 8 and 6h on days 100, 121 and 199, respectively. This acclimation strategy favored the retention of increasingly higher densities of well-adapted microbial populations in the reactor. The increases in TBA loading produced better settling biomass and higher biomass concentrations with higher specific TBA biodegradation rates. Effluent TBA concentrations were consistently below the detection limit of 25 microgL(-1). The use of progressively shorter cycle times created selection pressures that fostered the self-immobilization of the reactor microorganisms into aerobic granules which first appeared on day 125. Specific TBA biodegradation rates in the granules followed the Haldane model for substrate inhibition, and peaked at 13.8 mgTBAgVSS(-1)h(-1) at a TBA concentration of 300 mgL(-1). Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes from granules sampled between days 220 and 247 confirmed the existence of a highly stable microbial community with members belonging to the alpha, beta and delta subdivisions of Proteobacteria and the Cytophaga-Flavobacteria-Bacteroides (CFB) group.     

Influence of substrate surface loading on the kinetic behaviour of aerobic granules

In the aerobic granular sludge reactor, the substrate loading is related to the size of the aerobic granules cultivated. This study investigated the influence of substrate surface loading on the growth and substrate-utilization kinetics of aerobic granules. Results showed that microbial surface growth rate and surface biodegradation rate are fairly related to the substrate surface loading by the Monod-type equation. In this study, both the theoretical maximum growth yield and the Pirt maintenance coefficient were determined. It was found that the estimated theoretical maximum growth yield of aerobic granules was as low as 0.2 g biomass g^–1 chemical oxygen demand (COD) and 10–40% of input substrate-COD was consumed through the maintenance metabolism, while experimental results further showed that the unit oxygen uptake by aerobic granules was 0.68 g oxygen g^–1 COD, which was much higher than that reported in activated sludge processes. Based on the growth yield and unit oxygen uptake determined, an oxidative assimilation equation of acetate-fed aerobic granules was derived; and this was confirmed by respirometric tests. In aerobic granular culture, about 74% of the input substrate-carbon was converted to carbon dioxide. The growth yield of aerobic granules was three times lower than that of activated sludge. It is likely that high carbon dioxide production is the main cause of the low growth yield of aerobic granules, indicating a possible energy uncoupling in aerobic granular culture.