Visualization of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (x 1,000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, kappa=0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36-78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls=8.6, p=0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.     

Detection of terminal mismatches on DNA duplexes in homogeneous assays or with immobilized probes

We recently reported the design of new fluorescent oligo-2'-deoxyribonucleotides (FODNs) for the detection of terminal mismatches on DNA duplexes in homogeneous assays. We now report the validation of this method in homogeneous assays with other sequences and the feasibility of the detection of terminal mismatches with immobilized FODNs. In all cases studied, the mismatched duplexes were more fluorescent than the perfect ones and results confirmed that the discrimination factor is sequence-dependent.     

Stress-induced morphogenic responses: growing out of trouble?

Plants exposed to sub-lethal abiotic stress conditions exhibit a broad range of morphogenic responses. Despite the diversity of phenotypes, a generic 'stress-induced morphogenic response' can be recognized that appears to be carefully orchestrated and comprises three components: (a) inhibition of cell elongation, (b) localized stimulation of cell division and (c) alterations in cell differentiation status. It is hypothesized that the similarities in the morphogenic responses induced by distinct stresses, reflect common molecular processes such as increased ROS-production and altered phytohormone transport and/or metabolism. The stress-induced morphogenic response (SIMR) is postulated to be part of a general acclimation strategy, whereby plant growth is redirected to diminish stress exposure.     

Co-occurrence analysis of insertional mutagenesis data reveals cooperating oncogenes

MOTIVATION: Cancers are caused by an accumulation of multiple independent mutations that collectively deregulate cellular pathways, e.g. such as those regulating cell division and cell-death. The publicly available Retroviral Tagged Cancer Gene Database (RTCGD) contains the data of many insertional mutagenesis screens, in which the virally induced mutations result in tumor formation in mice. The insertion loci therefore indicate the location of putative cancer genes. Additionally, the presence of multiple independent insertions within one tumor hints towards a cooperation between the insertionally mutated genes. In this study we focus on the detection of statistically significant co-mutations. RESULTS: We propose a two-dimensional Gaussian Kernel Convolution method (2DGKC), a computational technique that identifies the cooperating mutations in insertional mutagenesis data. We define the Common Co-occurrence of Insertions (CCI), signifying the co-mutations that are statistically significant across all different screens in the RTCGD. Significance estimates are made on multiple scales, and the results visualized in a scale space, thereby providing valuable extra information on the putative cooperation. The multidimensional analysis of the insertion data results in the discovery of 86 statistically significant co-mutations, indicating the presence of cooperating oncogenes that play a role in tumor development. Since oncogenes may cooperate with several members of a parallel pathway, we combined the co-occurrence data with gene family information to find significant cooperations between oncogenes and families of genes. We show, for instance, the interchangeable cooperation of Myc insertions with insertions in the Pim family. AVAILABILITY: A list of the resulting CCIs is available at: http://ict.ewi.tudelft.nl/~jeroen/CCI/CCI_list.txt.     

Impact of carbon metabolism on 13C signatures of cyanobacteria and green non-sulfur-like bacteria inhabiting a microbial mat from an alkaline siliceous hot spring in Yellowstone National Park (USA)

Alkaline siliceous hot spring microbial mats in Yellowstone National Park are composed of two dominant phototropic groups, cyanobacteria and green non-sulfur-like bacteria (GNSLB). While cyanobacteria are thought to cross-feed low-molecular-weight organic compounds to support photoheterotrophic metabolism in GNSLB, it is unclear how this could lead to the heavier stable carbon isotopic signatures in GNSLB lipids compared with cyanobacterial lipids found in previous studies. The two groups of phototrophs were separated using percoll density gradient centrifugation and subsequent lipid and stable carbon isotopic analysis revealed that we obtained fractions with a approximately 60-fold enrichment in cyanobacterial and an approximately twofold enrichment in GNSLB biomass, respectively, compared with the mat itself. This technique was used to study the diel cycling and 13C content of the glucose pools in and the uptake of 13C-bicarbonate by the cyanobacteria and GNSLB, as well as the transfer of incorporated 13C from cyanobacteria to GNSLB. The results show that cyanobacteria have the highest bicarbonate uptake rates and accumulate glucose during the afternoon in full light conditions. In contrast, GNSLB have relatively higher bicarbonate uptake rates compared with cyanobacteria in the morning at low light levels. During the night GNSLB take up carbon that is likely derived through fermentation of cyanobacterial glucose enriched in 13C. The assimilation of 13C-enriched cyanobacterial carbon may thus lead to enriched 13C-contents of GNSLB cell components.     

Methane oxidation coupled to oxygenic photosynthesis in anoxic waters

Freshwater lakes represent large methane sources that, in contrast to the Ocean, significantly contribute to non-anthropogenic methane emissions to the atmosphere. Particularly mixed lakes are major methane emitters, while permanently and seasonally stratified lakes with anoxic bottom waters are often characterized by strongly reduced methane emissions. The causes for this reduced methane flux from anoxic lake waters are not fully understood. Here we identified the microorganisms and processes responsible for the near complete consumption of methane in the anoxic waters of a permanently stratified lake, Lago di Cadagno. Interestingly, known anaerobic methanotrophs could not be detected in these waters. Instead, we found abundant gamma-proteobacterial aerobic methane-oxidizing bacteria active in the anoxic waters. In vitro incubations revealed that, among all the tested potential electron acceptors, only the addition of oxygen enhanced the rates of methane oxidation. An equally pronounced stimulation was also observed when the anoxic water samples were incubated in the light. Our combined results from molecular, biogeochemical and single-cell analyses indicate that methane removal at the anoxic chemocline of Lago di Cadagno is due to true aerobic oxidation of methane fuelled by in situ oxygen production by photosynthetic algae. A similar mechanism could be active in seasonally stratified lakes and marine basins such as the Black Sea, where light penetrates to the anoxic chemocline. Given the widespread occurrence of seasonally stratified anoxic lakes, aerobic methane oxidation coupled to oxygenic photosynthesis might have an important but so far neglected role in methane emissions from lakes.     

Auxin transport at cellular level: new insights supported by mathematical modelling

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.     

Refined identification of neutralization-resistant HIV-1 CRF02_AG viruses

We studied neutralization of CRF02_AG HIV-1-infected plasma samples. In contrast to previous reports, these samples neutralized CRF02_AG viruses better than other viruses. This included six of eight CRF02_AG viruses previously designated resistant (tier 2/3 or 3). Only viruses 253-11 and 278-50 remained highly resistant, but they were sensitive to membrane-proximal external region (MPER)-specific monoclonal antibodies, suggesting neutralization targets for even these viruses. We propose using high-neutralizing-within-subtype samples for evaluation of neutralization resistance of viruses.