DNA stable-isotope probing

Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.     

Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker

Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene degradation and has been found in all anaerobic toluene degrading bacterial isolates tested. However, only a few pure cultures capable of anaerobic toluene oxidation are available to date, and it is important to understand the relevance of these model organisms for in situ bioremediation of hydrocarbon-contaminated aquifers. Due to their phylogenetic dispersal, it is not possible to specifically target anaerobic toluene degraders using marker rRNA genes. We therefore established an assay targeting a approximately 794 bp fragment within the Bss alpha-subunit (bssA) gene, which allows for the specific detection and affiliation of both known and unknown anaerobic degraders. Three distinct tar-oil-contaminated aquifer sites were screened for intrinsic bssA gene pools in order to identify and compare the diversity of hydrocarbon degraders present at these selected sites. We were able to show that local diversity patterns of degraders were entirely distinct, apparently highly specialized and well-adapted to local biogeochemical settings. Discovered at one of the sites were bssA genes closely related to that of Geobacter spp., which provides evidence for an importance of iron reduction for toluene degradation in these sediments. Retrieved from the other two sites, dominated by sulfate reduction, were previously unidentified bssA genes and also deeply branching putative bssA homologues. We provide evidence for a previously unrecognized diversity of anaerobic toluene degraders and also of other hydrocarbon degraders using fumarate-adding key reactions in contaminated aquifers. These findings enhance our current understanding of intrinsic hydrocarbon-degrading microbial communities in perturbed aquifers and may have potential for the future assessment and prediction of natural attenuation based on degradation genes.     

13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea

Marine pelagic redoxclines are zones of high dark CO₂ fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO₂ fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO₂ incorporation in ¹³C-bicarbonate pulse experiments. The incorporation of ¹³C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in ¹³C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO₂-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the ¹³C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO₂-fixing Proteobacteria within this habitat.     

Young patients with colorectal cancer have poor survival in the first twenty months after operation and predictable survival in the medium and long-term: Analysis of survival and prognostic markers

Introduction

Male breast cancer (MBC) is a rare, yet potentially aggressive disease. Although literature regarding female breast cancer (FBC) is extensive, little is known about the etiopathogenesis of male breast cancer. Studies from our laboratory show that MBCs have a distinct immunophenotypic profile, suggesting that the etiopathogenesis of MBC is different from FBCs. The aim of this study was to evaluate and correlate the immunohistochemical expression of cell cycle proteins in male breast carcinoma to significant clinico-biological endpoints.

Methods

75 cases of MBC were identified using the records of the Saskatchewan Cancer Agency over 26 years (1970-1996). Cases were reviewed and analyzed for the immunohistochemical expression of PCNA, Ki67, p27, p16, p57, p21, cyclin-D1 and c-myc and correlated to clinico-biological endpoints of tumor size, node status, stage of the disease, and disease free survival (DFS).

Results

Decreased DFS was observed in the majority of tumors that overexpressed PCNA (98%, p = 0.004). The overexpression of PCNA was inversely correlated to the expression of Ki67 which was predominantly negative (78.3%). Cyclin D1 was overexpressed in 83.7% of cases. Cyclin D1 positive tumors were smaller than 2 cm (55.6%, p = 0.005), had a low incidence of lymph node metastasis (38.2%, p = 0.04) and were associated with increased DFS of >150 months (p = 0.04). Overexpression of c-myc (90%) was linked with a higher incidence of node negativity (58.3%, p = 0.006) and increased DFS (p = 0.04). p27 over expression was associated with decreased lymph node metastasis (p = 0.04). P21 and p57 positive tumors were related to decreased DFS (p = 0.04). Though p16 was overexpressed in 76.6%, this did not reach statistical significance with DFS (p = 0.06) or nodal status (p = 0.07).

Conclusion

Aberrant cell cycle protein expression supports our view that these are important pathways involved in the etiopathogenesis of MBC. Tumors with overexpression of Cyclin D1 and c-myc had better outcomes, in contrast to tumors with overexpression of p21, p57, and PCNA with significantly worse outcomes. P27 appears to be a predictive marker for lymph nodal status. Such observation strongly suggests that dysregulation of cell cycle proteins may play a unique role in the initiation and progression of disease in male breast cancer. Such findings open up new avenues for the treatment of MBC as a suitable candidate for novel CDK-based anticancer therapies in the future.     

Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Mapping of mouse intracisternal A-particle proviral markers in an interspecific backcross

We present a linkage map of intracisternal A-particle (IAP) proviral loci. The IAP family consists of 2000 endogenous proviral elements that are widely dispersed in the mouse genome. The map was constructed by using an interspecific backcross and markers defined by oligonucleotide probes specific for subclasses of expressed IAP elements. In genomic DNA from C57BL/6J mouse, these probes each detected from 12 to 44 HindIII restriction fragments that represent junctions between proviral and 5-flanking DNA. The fragments have characteristic strain distribution patterns (SDPs) that are particularly polymorphic in the DNAs of C57BL/6J and Mus spretus mice used for the backcross. IAP loci were placed on the map by comparison of their distribution patterns with those of known genetic markers in the backcross. The map includes 51 IAP loci that have not been previously mapped and 23 IAP proviruses that had been previously mapped in recombinant inbred (RI) strains. Comparable map positions were obtained with the IAP markers in the interspecific backcross and the RI strains. The mapped IAP loci were widely dispersed on the X Chromosome (Chr) and all of the autosomes except Chrs 9 and 19, providing useful genetic markers for linkage studies.     

Differential response of type C and intracisternal type A particle markers in cells treated with iododeoxyuridine and dexamethasone.

Mouse neuroblastoma cells containing intracisternal type A particles were treated with iododeoxyuridine and dexamethasone to induce the release of type C oncornavirus particles. For 5 days after treatment, antigenic markers and DNA polymerase activities specific to particles of each of the two types were assayed in the cells and in pellets obtained by high-speed centrifugation of the culture fluid. There was a marked release of C-particle antigen (p30) and DNA polymerase activity in extracellular particulate form, reaching a maximum on day 3 after treatment and falling thereafter. In contrast, no extracellular A-particle antigen was detected, and A-particle-specific DNA polymerase activity in the medium pellets did not increase from the original very low level. Electron microscopy confirmed the presence of free type C virus particles, but not intracisternal type A particles, in the culture fluid. Although intracellular levels of C-particle antigen rose 20- to 30-fold per milligram of cell protein, intracellular A-particle antigen and DNA polymerase activity did not vary more than two-fold. The relative rate of A-particle synthesis in the treated cells, as judged by incorporation of radioactive amino acids into the major structural protein (P73), was also unchanged over the period of observation. Thus, the induction of type C virus particle formation in cultured neuroblastoma cells had no detectable effect on the quantity, synthesis rate, or location of intracisternal type A particles.     

A short interspersed repetitive element found near some mouse structural genes.

We have isolated and characterized a family of interspersed repetitive elements which make up about 1% of the mouse genome. The elements represent a group of homologous but non-identical units about 400 bp in length. Individual members of the family show considerable divergence from one another. The spacial relationships between members of the family and a number of other identified mouse sequences including structural genes have been determined; these elements are found on the 5' as well as 3' sides of various genes at distances ranging from less than 1 to 7.5 kilobases (Kb). The sequences are present in the DNA of all species of Mus. Related sequences are present in the rat genome at a repetition frequency similar to that in the mouse genome. A partial sequence of one member of the family is presented.