Combining catalyzed reporter deposition-fluorescence in situ hybridization and microautoradiography to detect substrate utilization by bacteria and Archaea in the deep ocean

The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4',6'-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up l-aspartic acid (19% versus 10%).     

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor''s response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in α-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.     

New Colors for Histology: Optimized Bivariate Color Maps Increase Perceptual Contrast in Histological Images

Background

Accurate evaluation of immunostained histological images is required for reproducible research in many different areas and forms the basis of many clinical decisions. The quality and efficiency of histopathological evaluation is limited by the information content of a histological image, which is primarily encoded as perceivable contrast differences between objects in the image. However, the colors of chromogen and counterstain used for histological samples are not always optimally distinguishable, even under optimal conditions.

Methods and Results

In this study, we present a method to extract the bivariate color map inherent in a given histological image and to retrospectively optimize this color map. We use a novel, unsupervised approach based on color deconvolution and principal component analysis to show that the commonly used blue and brown color hues in Hematoxylin—3,3’-Diaminobenzidine (DAB) images are poorly suited for human observers. We then demonstrate that it is possible to construct improved color maps according to objective criteria and that these color maps can be used to digitally re-stain histological images.

Validation

To validate whether this procedure improves distinguishability of objects and background in histological images, we re-stain phantom images and N = 596 large histological images of immunostained samples of human solid tumors. We show that perceptual contrast is improved by a factor of 2.56 in phantom images and up to a factor of 2.17 in sets of histological tumor images.

Context

Thus, we provide an objective and reliable approach to measure object distinguishability in a given histological image and to maximize visual information available to a human observer. This method could easily be incorporated in digital pathology image viewing systems to improve accuracy and efficiency in research and diagnostics.     

Microbial load of drinking water reservoir tributaries during extreme rainfall and runoff

Hygienic and microbiological examinations of watercourses are usually not carried out during heavy rainfall and runoff events. After rainfall or snowmelt, there are often massive increases in turbidity in flooding creeks in mountain ranges, which are frequently interpreted as an indication of microbial contamination. The aim of this study was to quantify the microbial loads of watercourses during such runoff events and to compare these loads with loads occurring during regular conditions. In a 14-month monitoring period we investigated the microbial loads of three tributaries of different drinking water reservoirs. A total of 99 water samples were taken under different runoff conditions and analyzed to determine physical, chemical, bacterial, and parasitic parameters. Thirty-two water samples were considered event samples during nine measuring series. The criteria for events, based on duration and intensity of precipitation, water depth gauge measurements, and dynamics, had been fixed before the investigation for each creek individually. Of the physical and chemical parameters examined, only the turbidity, pH, and nitrate values differed clearly from the values obtained for regular samples. Most of the bacteriological parameters investigated (colony, Escherichia coli, coliform, fecal streptococcal, and Clostridium perfringens counts) increased considerably during extreme runoff events. If relevant sources of parasitic contamination occurred in catchment areas, the concentrations of Giardia and Cryptosporidium rose significantly during events. The results show that substantial shares of the total microbial loads in watercourses and in drinking water reservoirs result from rainfall and extreme runoff events. Consequently, regular samples are considered inadequate for representing the microbial contamination of watercourse systems. The procedures for raw water surveillance in the context of multiple-barrier protection and risk assessment ought to include sampling during extreme runoff situations.     

Hyperbaric oxygen induces apoptosis via a mitochondrial mechanism

During therapeutic hyperbaric oxygenation lymphocytes are exposed to high partial pressures of oxygen. This study aimed to analyze the mechanism of apoptosis induction by hyperbaric oxygen. For intervals of 0.5–4 h Jurkat-T-cells were exposed to ambient air or oxygen atmospheres at 1–3 absolute atmospheres. Apoptosis was analyzed by phosphatidylserine externalization, caspase-3 activation and DNA-fragmentation using flow cytometry. Apoptosis was already induced after 30 min of hyperbaric oxygenation (HBO, P < 0.05). The death receptor Fas was downregulated. Inhibition of caspase-9 but not caspase-8 blocked apoptosis induction by HBO. Hyperbaric oxygen caused a loss of mitochondrial membrane potential and caspase-9 induction. The mitochondrial pro-survival protein Bcl-2 was upregulated, and antagonizing Bcl-2 function potentiated apoptosis induction by HBO. In conclusion, a single exposure to hyperbaric oxygenation induces lymphocyte apoptosis by a mitochondrial and not a Fas-related mechanism. Regulation of Fas and Bcl-2 may be regarded as protective measures of the cell in response to hyperbaric oxygen.     

Effect of fire on the topsoil seed banks of rehabilitated bauxite mine sites in the jarrah forest of Western Australia

Summary Germinable seed stores of 5- and 8-year-old rehabilitated bauxite mine pits in south-west Western Australia were assessed before and after burning. These seed stores were compared to those of adjacent unmined Jarrah ( Eucalyptus marginata ) forest, to identify at what age fire can be reintroduced, in order to measure restoration success and reduce fire hazard. Soils were sampled in early summer (before fire) and late autumn (after fire). Before fire, the mean topsoil seed bank of 5-year-old sites was 2121 seeds per m² while 8-year-old sites had a mean of 1520 seeds per m². Only the 5-year-old sites were significantly different from the forest mean of 1478 seeds per m² for the same season. After summer burns (and possibly due to seasonal effects) topsoil seed banks of rehabilitated areas (sampled in autumn) decreased by an average of 53 per cent. Topsoil seed banks of 5–8-year-old sites were resistant to lower intensity burns, with 362 seeds per m² of native species surviving mild burns and 108 seeds per m² of native species surviving after an intense summer fire. The topsoil seed reserve of 5–8-year-old rehabilitated areas had a high proportion of annual weed species while the forest sites had high levels of subshrubs and native annuals. Low-intensity burns did not alter the composition of life-forms in the soil seed bank, while intense burns favoured annual weed and shrub species. The results indicate that it is not appropriate to introduce fire to rehabilitated areas before 8 years, due to limited fuel reduction benefits and possible adverse effects on obligate seeding species. The large proportion of weed species in the soil seed bank of young rehabilitated areas is a concern, and remains a major consideration for future disturbance of these areas.     

Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.     

Reduced apo-fumarate nitrate reductase regulator (apoFNR) as the major form of FNR in aerobically growing Escherichia coli

Under anoxic conditions, the Escherichia coli oxygen sensor FNR (fumarate nitrate reductase regulator) is in the active state and contains a [4Fe-4S] cluster. Oxygen converts [4Fe-4S]FNR to inactive [2Fe-2S]FNR. After prolonged exposure to air in vitro, apoFNR lacking a Fe-S cluster is formed. ApoFNR can be differentiated from Fe-S-containing forms by the accessibility of the five Cys thiol residues, four of which serve as ligands for the Fe-S cluster. The presence of apoFNR in aerobically and anaerobically grown E. coli was analyzed in situ using thiol reagents. In anaerobically and aerobically grown cells, the membrane-permeable monobromobimane labeled one to two and four Cys residues, respectively; the same labeling pattern was found with impermeable thiol reagents after cell permeabilization. Alkylation of FNR in aerobic bacteria and counting the labeled residues by mass spectrometry showed a form of FNR with five accessible Cys residues, corresponding to apoFNR with all Cys residues in the thiol state. Therefore, aerobically growing cells contain apoFNR, whereas a significant amount of Fe-S-containing FNR was not detected under these conditions. Exposure of anaerobic bacteria to oxygen caused conversion of Fe-S-containing FNR to apoFNR within 6 min. ApoFNR from aerobic bacteria contained no disulfide, in contrast to apoFNR formed in vitro by air inactivation, and all Cys residues were in the thiol form.