Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼ 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼ 10^− 4 of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T = 7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼ 15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (< 28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.     

Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement for monitoring programs. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins. Assays are based on the discrimination of genetic differences in the species. A commercially available PCR ELISA Dig Detection Kit in a microtiter plate was adapted for the rapid assessment of specificity of the two probes used in a sandwich hybridization assay. The toxic dinoflagellate Alexandrium minutum was used as the target organism and a capture and signal probe were designed for a species-specific identification of this species. This assay also provided the necessary specificity tests prior to the probes being adapted to an automated biosensor using a sandwich hybridization format. All probes regardless of the detection method must be extensively tested prior to use in the field. Total rRNA was isolated from three different strains of A. minutum and the mean concentration of RNA per cell of was determined to be 0.028 ng ± 0.003. Thus, a standard calibration curve for different RNA concentrations was determined so that cell numbers could be inferred from the assay. The assay and the standard curve were evaluated by using spiked field samples. The results demonstrated that the molecular assay was able to detect A. minutum cells at different cell counts in the presence of a complex background.     

Terrestrial origin of bacterial communities in complex boreal freshwater networks

Bacteria inhabiting boreal freshwaters are part of metacommunities where local assemblages are often linked by the flow of water in the landscape, yet the resulting spatial structure and the boundaries of the network metacommunity have never been explored. Here, we reconstruct the spatial structure of the bacterial metacommunity in a complex boreal aquatic network by determining the taxonomic composition of bacterial communities along the entire terrestrial/aquatic continuum, including soil and soilwaters, headwater streams, large rivers and lakes. We show that the network metacommunity has a directional spatial structure driven by a common terrestrial origin of aquatic communities, which are numerically dominated by taxa recruited from soils. Local community assembly is driven by variations along the hydrological continuum in the balance between mass effects and species sorting of terrestrial taxa, and seems further influenced by priority effects related to the spatial sequence of entry of soil bacteria into the network.     

Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants.

Toxoplasma gondii, strain RH, produced plaques in human fibroblast tissue cultures over the temperatures 30–41 C. Muta?enesis with N-methyl-N′-nitro-N-nitrosoguanidine yielded seven temperature-sensitive mutants that had lost the ability to form plaques at 40 C but still grew well at 33 C. No spontaneous mutants were detected. The temperature-sensitive mutants were not markedly thermolabile and adsorbed normally to tissue culture cells at 40 C. Three mutants differed from one another in their temperatures for optimal growth, and in their ability to remain infectious within cells incubated at 40 C. Both mutants that were tested were found to be markedly less virulent for mice than was the wild type RH strain.     

Automated Assay of Telomere Length Measurement and Informatics for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort

The Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort includes DNA specimens extracted from saliva samples of 110,266 individuals. Because of its relationship to aging, telomere length measurement was considered an important biomarker to develop on these subjects. To assay relative telomere length (TL) on this large cohort over a short time period, we created a novel high throughput robotic system for TL analysis and informatics. Samples were run in triplicate, along with control samples, in a randomized design. As part of quality control, we determined the within-sample variability and employed thresholds for the elimination of outlying measurements. Of 106,902 samples assayed, 105,539 (98.7%) passed all quality control (QC) measures. As expected, TL in general showed a decline with age and a sex difference. While telomeres showed a negative correlation with age up to 75 years, in those older than 75 years, age positively correlated with longer telomeres, indicative of an association of longer telomeres with more years of survival in those older than 75. Furthermore, while females in general had longer telomeres than males, this difference was significant only for those older than age 50. An additional novel finding was that the variance of TL between individuals increased with age. This study establishes reliable assay and analysis methodologies for measurement of TL in large, population-based human studies. The GERA cohort represents the largest currently available such resource, linked to comprehensive electronic health and genotype data for analysis.     

A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.     

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high‐abundant serum proteins by partial denaturation and enrichment of low‐abundant biomarkers by size exclusion chromatography. The recovery of low‐abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody‐based strategies, and offers the possibility of multiplexing. Our proof‐of‐principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Effect of the phytoestrogen, genistein-8-C-glucoside, on Chinese hamster ovary cells in vitro

Genistein-8-C-glucoside (G8CG) belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. A number of studies on flavonoids show their cardioprotective and antiosteoporosis properties in in vitro and in vivo models. As a phytoestrogen, genistein has recently generated interest as a potential anticancer and antiatherogenic agent. Several flavonoids are known as antioxidants and scavengers of free oxygen radicals. In the current investigation we used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.). Many authors have found that the action of genistein is not so simple, although many reports conducted in vitro have demonstrated that it is cytotoxic and genotoxic. Therefore, the cytotoxic and genotoxic effects of this compound in Chinese hamster ovary cells (line CHO) were studied. A colorimetric MTT assay to assess cytotoxicity and a Comet assay for the detection of DNA damage were used. Apoptosis was determined by the Hoechst 33258/propidium iodide staining technique. We have also demonstrated antioxidant properties of G8CG. The level of reactive oxygen species generated by G8CG alone and/or H2O2 was evaluated with fluorescence probes: dichlorofluorescein-diacetate (DCFDA) by flow cytometry. The cells were exposed to various concentrations of genistein-8-C-glucoside (1-290 microM) and hydrogen peroxide (10-130 microM) and the effect of G8CG alone or in combination with H2O2 was determined. The results reveal that G8CG at concentrations higher than 10 microM significantly reduced cell viability, induced apoptosis and DNA damage. However at lower concentrations (5 and 7.5 microM), G8CG showed antioxidant properties, but had no cytotoxic or genotoxic activity.     

Canine distemper virus utilizes different receptors to infect chicken embryo fibroblasts and vero cells

Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts (CEF) is a common method to develop attenuated live vaccines with full security. Canine distemper virus (CDV) also does this, but the mechanisms and particular receptors remain unclear. Virus overlay protein blot assays were carried out on CEF membrane proteins, which were extracted respectively with a Mem-PER™ kit, a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method, and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells, indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.