Concentration-dependent response of estrone-degrading bacterial community in activated sludge analyzed by microautoradiography-fluorescence in situ hybridization

Inefficient removal of estrone (E1) in wastewater treatment plants (WWTPs) causes feminizing effects in male aquatic creatures. As E1 is mainly removed by biodegradation, investigation of E1 degradation is important to determine better removal strategies. Using microautoradiography-fluorescence in situ hybridization (MAR-FISH), we demonstrated that the structures of [³H]E1-incorporating bacterial communities were different at different E1 concentrations applied to activated sludge. At 200 μg/L E1, almost all [³H]E1-incorporating cells were associated with either Betaproteobacteria or Gammaproteobacteria (60% and 40% of MAR (+) cells, respectively). The proportion of Betaproteobacteria and Gammaproteobacteria in the total number of [³H]E1-incorporating cells decreased as the concentration of E1 decreased. In contrast, the proportion of Alphaproteobacteria in the total number of [³H]E1-incorporating cells increased as the concentrations of E1 decreased. At the lowest applied concentration (540 ng/L), almost all the [³H]E1-incorporating cells were Alphaproteobacteria (96%). The results of MAR-FISH applied to sludge samples collected from various plant locations and activated sludge processes, and during different seasons also demonstrated the high contribution of Alphaproteobacteria to the entire E1-degrading bacterial community (50.4 ± 11% of the total number of [³H]E1-incorporating cells) at 1 μg/L E1. Since the E1 concentration in domestic wastewater is at sub-μg/L levels, the key E1 degraders in activated sludge of domestic WWTPs are probably be Alphaproteobacteria. All [³H]E1-incorporating Alphaproteobacteria were hybridized with probe ALF968. Few MAR (+) cells were Sphingomonadales. An E1-degrading bacterial community at low E1 concentration appeared to consist of diverse bacterial groups of Alphaproteobacteria. This study suggested that substrate concentration is an essential factor for revealing E1-degrading bacteria in complex communities.     

Identification of chicken-specific fecal microbial sequences using a metagenomic approach

In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ among different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (CP) and between chicken fecal DNA and an avian DNA composite consisting of turkey, goose, and seagull fecal DNA extracts (CB) to enrich for chicken-specific DNA fragments. A total of 471 non-redundant chicken metagenomic sequences were retrieved and analyzed. All of the clone sequences were similar to prokaryotic genes, of which more than 60% could not be assigned to previously characterized functional roles. In general terms, sequences assigned characterized functional roles were associated with cellular processes (11.7%), metabolism (11.0%) and information storage and processing (13.4%). Approximately 53% of the non-redundant sequences are similar to genes present in intestinal bacteria belonging to Clostridia (20.9%), Bacteroidetes (15.0%), and Bacilli (17.3%). Twenty-five sequences from the CP and CB clone libraries were selected to develop chicken fecal-specific PCR assays. These assays were challenged against fecal DNA extracted from 21 different animal species, including mammals and birds. The results from the host-specificity studies showed that 12 of the assays had a high degree of specificity to chicken feces. In addition, three assays were specific to chicken and turkey while another four assays tested positive to more than two avian species, suggesting a broader distribution of some of the enriched gene fragments among different avian fecal microbial communities. Fecal pollution signals were detected using chicken-specific assays in contaminated water samples, although the PCR assays showed different detection limits. These results indicate the need for multiple assays to detect poultry fecal sources of pollution. The competitive DNA hybridization approach used in this study can rapidly select for numerous chicken fecal metagenomic regions that can be used as potential genetic markers for fecal source tracking.     

葡萄属(Vitis)种间杂交一代(F_1)果实抗白粉病的遗传

通过对７个欧洲葡萄品种与４个中国葡萄野生种组成的１１个杂交组合的果实抗白粉病田间自然发病调查，结果表明，后代群体的抗病性呈连续性分布，表现出多基因遗传的特征。在供试的中国葡萄资源中，华东葡萄、毛葡萄和秋葡萄具有将其抗性遗传给后代（Ｆ１）的较强能力。     

花粉管通道法及其在烟草育种中的应用综述

对远缘杂交的主要方法 :常规有性杂交、体细胞杂交、离体受精、花粉管通道、基因转导进行了比较 ,并探讨了花粉管通道法在烟草育种中的应用前景     

Enterotoxigenic Escherichia coli detected in foods by PCR and an enzyme-linked oligonucleotide probe

A polymerase chain reaction (PCR) and an enzyme-linked oligonucleotide probe hybridization assay were developed for the detection of enterotoxigenic Escherichia coli (ETEC) in ground beef, chicken, pork and raw milk. Two synthetic primers, one of which was biotinylated, were used in the PCR to amplify a fragment of the E. coli heat-labile enterotoxin (LT) gene. The identity of the amplified products was confirmed by liquid hybridization using a horseradish peroxidase-linked internal oligonucleotide probe in a 96-well microplate coated with streptavidin. The final quantitation of the PCR products was performed by a colorimetric reaction. Under established conditions (including l min at 60 °C for primer annealing and extension in PCR cycles), this method detected all 7 LT-producing E. coli pathogenic for humans, but did not detect all 7 LT-positive E. coli of animal origin, 3 E. coli strains that do not produce LT, and 9 other bacteria. Under less stringent PCR conditions (55 °C for annealing and extension), 2 strains of LT-producing E. coli of porcine origin were detected while the results of other bacterial strains remained unchanged. In pure cultures, the detection limit of the method was 1.4 colony forming units (CFU). Prior to PCR amplification, all food samples inoculated with an LT-producing ETEC, were subjected to enrichment in brain heart infusion broth for 8 h at 37 °C. From these cultures, 10 μ1 was heated at 95 °C for 10 min and directly used in the PCR. An initial inoculum of as few as 1.2 to 12 CFU of the LT-producing ETEC per 25 g (or ml) of food sample gave a positive reaction.     

Intracellular localization of dietary and naked DNA in intestinal tissue of Atlantic salmon, Salmo salar L. using in situ hybridization

There is a continuing interest in the fate of DNA from genetically modified organisms (GMO) in the food chain including the uptake of DNA by intestinal cells from dietary sources containing GM feed ingredients. The objective of this study was to elucidate the uptake and persistence of foreign DNA in the intestinal tract of Atlantic salmon Salmo salar L. using in situ hybridization (ISH) that enables the intracellular localization of the DNA, and polymerase chain reaction (PCR) to verify the ISH results qualitatively. Two salmon intestinal models were employed for the investigations; intestinal tissues were sampled in two models namely (a) in vivo from salmon-fed diets containing 30% GM soybeans or 30% nonGM (nGM) soybeans, and (b) ex vivo from intestinal sleeves incubated using different concentrations of PCR-amplified test DNAs (211 and 305 bp) designed from the 35S promoter/plant DNA junction of the RoundupReady soybean (RRS) genome. Additionally, for the incubation study, the effect of a mucolytic agent dithiothreitol (DTT) and a permeability enhancer sodium deoxycholate (SDA) on DNA uptake were investigated. Both treatments were found to enhance DNA uptake ex vivo. Dietary DNA and PCR-amplified DNA could be visualized by ISH in the salmon intestine with more frequently observed signals in the ex vivo model compared to the in vivo model. All results could be verified by PCR. Dietary DNA was localized in the cell vacuolar system and in lamina propria of the mid intestine. Thus, based on the investigated DNA fragment lengths, this study shows that foreign DNA, can be taken up by Atlantic salmon intestinal tissue.     

Analysis of Photoelectric Hybridization Measurementon Shearing Force for Flat Shearer

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Ultra‐Specific Zeptomole MicroRNA Detection by Plasmonic Nanowire Interstice Sensor with Bi‐Temperature Hybridization

MicroRNAs (miRNAs) are emerging new biomarkers for many human diseases. To fully employ miRNAs as biomarkers for clinical diagnosis, it is most desirable to accurately determine the expression patterns of miRNAs. The optimum miRNA profiling method would feature 1) highest sensitivity with a wide dynamic range for accurate expression patterns, 2) supreme specificity to discriminate single nucleotide polymorphisms (SNPs), and 3) simple sensing processes to minimize measurement variation. Here, an ultra‐specific detection method of miRNAs with zeptomole sensitivity is reported by applying bi‐temperature hybridizations on single‐crystalline plasmonic nanowire interstice (PNI) sensors. This method shows near‐perfect accuracy of SNPs and a very low detection limit of 100 am (50 zeptomole) without any amplification or labeling steps. Furthermore, multiplex sensing capability and wide dynamic ranges (100 am –100 pm ) of this method allows reliable observation of the expression patterns of miRNAs extracted from human tissues. The PNI sensor offers combination of ultra‐specificity and zeptomole sensitivity while requiring two steps of hybridization between short oligonucleotides, which could present the best set of features for optimum miRNA sensing method.