Microscale distribution of populations and activities of Nitrosospira and Nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 microm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.     

Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control

Histone deacetylases (HDACs) are enzymes that modify key residues in histones to regulate chromatin architecture, and they play a vital role in cell survival, cell-cycle progression, and tumorigenesis. To understand the function of Hdac3, a critical component of the N-CoR/SMRT repression complex, a conditional allele of Hdac3 was engineered. Cre-recombinase-mediated inactivation of Hdac3 led to a delay in cell-cycle progression, cell-cycle-dependent DNA damage, and apoptosis in mouse embryonic fibroblasts (MEFs). While no overt defects in mitosis were observed in Hdac3-/- MEFs, including normal H3Ser10 phosphorylation, DNA damage was observed in Hdac3-/- interphase cells, which appears to be associated with defective DNA double-strand break repair. Moreover, we noted that Hdac3-/- MEFs were protected from DNA damage when quiescent, which may provide a mechanistic basis for the action of HDAC inhibitors on cycling tumor cells.     

A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones

The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes. Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S-23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9-99.4% sequence identity). This cluster showed only 相似文献   

Therapeutic window of an EpCAM/CD3-specific BiTE antibody in mice is determined by a subpopulation of EpCAM-expressing lymphocytes that is absent in humans

MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that muS110 has significant anti tumor activity at well-tolerated doses as low as 5 μg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143–151, 2008). Here, we have explored the safety profile of muS110 at higher doses. Escalation to 50 μg/kg muS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-α, IL-6, IL-2, IFN-γ and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM⁺ cells. Various mouse strains presented with a subpopulation of 2–3% EpCAM⁺ blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM⁺ cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to muS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM⁺ lymphocytes in the observed side effects, reduction of EpCAM⁺ blood cells in mice via a low-dose pre treatment with muS110 dramatically increased the tolerability of animals up to at least 500 μg/kg of the BiTE antibody. This high tolerability to muS110 occurred in the presence of non-compromised T cells. No damage to EpCAM⁺ epithelial tissues was evident from histopathological examination of animals daily injected with 100 μg/kg muS110 for 28 days. In summary, these observations suggest that side effects of muS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM⁺ lymphocytes. Because humans have extremely low numbers of EpCAM⁺ cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice. M. Amann and M. Friedrich contributed equally to this work.     

Improved pre-concentration and detection methods for volatile sulphur breath constituents

Suitability of different types of pre-concentration (solid phase microextraction and sorbent trapping) and detection (flame photometric detector (FPD) and mass selective detector (MSD)) for gas chromatographic determination of sulphur-containing compounds (H₂S, MeSH, EtSH, DMS, COS and CS₂) in breath-gas was assessed in this study. Several factors like influence of humidity, influence of oxygen, or stability of target compounds in extraction vessels (SPME vials and sorbent tubes) were investigated. Despite poor stability of VSCs in SPME vials and matrix effects (unfavorable influence of humidity), SPME was found to be a fast and reliable enrichment method, which coupled with mass selective detector provided satisfactory LODs of target compounds at the ppt level (from 0.15 ppb for CS₂ to 2.3 ppb for H₂S). Application of sorbent trapping with two-bed sorbent tubes containing Tenax TA and Carboxen 1000 gave excellent LODs (0.03–0.3 ppb for 200 ml sample and MSD). Stability of investigated VSCs in sorbents was found to be very poor (30–40% losses after 2 h). FPD showed satisfactory sensitivity only when it was coupled with sorbent trapping. Breath samples were collected into Tedlar bags in a CO₂-controlled manner. Humidity was removed during sampling (permeation dryer – Nafion) to avoid unfavorable water dependent effects during analysis.     

Optimization of the dilute maleic acid pretreatment of wheat straw

Background  

In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.     

Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME‐1 group

Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate‐reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME‐1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82–90% of a composite ANME‐1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME‐1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron‐accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]‐hydrogenases are lacking in the dataset, genes for an [FeFe]‐hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c‐type cytochromes were identified, which have not yet been correlated with methanogenesis‐related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME‐1 to the bacterial sulfate‐reducing partner.