Microbial Labilization and Diversification of Pyrogenic Dissolved Organic Matter

With the increased occurrence of wildfires around the world, interest in the chemistry of pyrogenic organic matter (pyOM) and its fate in the environment has increased. Upon leaching from soils by rain events, significant amounts of dissolved pyOM (pyDOM) enter the aquatic environment and interact with microbial communities that are essential for cycling organic matter within the different biogeochemical cycles. To evaluate the biodegradability of pyDOM, aqueous extracts of laboratory-produced biochars were incubated with soil microbes, and the molecular changes to the composition of pyDOM were probed using ultrahigh-resolution mass spectrometry (Fourier transform–ion cyclotron resonance–mass spectrometry). Given that solar irradiation significantly affects the composition of pyDOM during terrestrial-to-marine export, the effects of photochemistry were also evaluated in the context of pyDOM biodegradability. Ultrahigh-resolution mass spectrometry revealed that many different (both aromatic and aliphatic) compounds were biodegraded. New labile compounds were produced, 22 %–40 % of which were peptide-like. These results indicated that a portion of pyDOM has been labilized into microbial biomass during the incubations. Fluorescence excitation–emission matrix spectra revealed that some fraction of these new bio-produced molecules is associated with proteinaceous fluorophores. Two-dimensional 1H–1H total correlation nuclear magnetic resonance (NMR) spectroscopy identified a peptidoglycan-like backbone within the microbially produced compounds. These results are consistent with previous observations of peptidoglycans within the soil and ocean nitrogen cycles where remnants of biodegraded pyDOM are expected to be observed. Interestingly, the exact nature of the bio-produced organic matter was found to vary drastically among samples indicating that the microbial consortium used may produce different exudates based on the composition of the initial pyDOM. Another potential explanation for the vast diversity of molecules is that microbes only consume low molecular-weight compounds, but they also produce reactive oxygen species (ROS), which initiate oxidative and recombination reactions that degrade high molecular-weight compounds and produce new molecules. Some of the bio-produced molecules (212–308 molecular formulas) were identified in estuarine and marine (surface and abyssal oceanic), and 81–192 of these formulas were of molecular composition attributed to carboxyl-rich alicyclic molecules (CRAM). These results indicate that some of the pyDOM biodegradation products have an oceanic fate and can be sequestered into the deep ocean. The observed microbially mediated diversification of pyDOM suggests that pyDOM contributes to the observed large complexity of natural organic matter observed in riverine and oceanic systems. More broadly, our research shows that pyDOM can be substrate for microbial growth and be incorporated into environmental food webs within the global carbon and nitrogen cycles

Integrated management of chronic kidney disease: Analysis of an innovative policy in Portugal

The implementation in Portugal of a model for integrated management of disease applied to the end-stage renal disease, from 2008 onwards, has completely restructured the way of providing care, as well as the payment and follow-up of patients under dialysis. This article describes the steps taken by the Ministry of Health, in terms of the planning, implementation and follow-up of the policy, with a particular focus on the importance of involving the various groups of interest, the leadership, as well as the capacity of negotiation and influence of the Government

Continuous and Long-Term Volume Measurements with a Commercial Coulter Counter

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter.National Institutes of Health (U.S.) (EUREKA Exceptional, Unconventional Research Enabling Knowledge Acceleration (R01GM085457))National Institutes of Health (U.S.) (contract R21CA137695)National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874

Control of Transcription by Cell Size

Cell size increases significantly with increasing ploidy. Differences in cell size and ploidy are associated with alterations in gene expression, although no direct connection has been made between cell size and transcription. Here we show that ploidy-associated changes in gene expression reflect transcriptional adjustment to a larger cell size, implicating cellular geometry as a key parameter in gene regulation. Using RNA-seq, we identified genes whose expression was altered in a tetraploid as compared with the isogenic haploid. A significant fraction of these genes encode cell surface proteins, suggesting an effect of the enlarged cell size on the differential regulation of these genes. To test this hypothesis, we examined expression of these genes in haploid mutants that also produce enlarged size. Surprisingly, many genes differentially regulated in the tetraploid are identically regulated in the enlarged haploids, and the magnitude of change in gene expression correlates with the degree of size enlargement. These results indicate a causal relationship between cell size and transcription, with a size-sensing mechanism that alters transcription in response to size. The genes responding to cell size are enriched for those regulated by two mitogen-activated protein kinase pathways, and components in those pathways were found to mediate size-dependent gene regulation. Transcriptional adjustment to enlarged cell size could underlie other cellular changes associated with polyploidy. The causal relationship between cell size and transcription suggests that cell size homeostasis serves a regulatory role in transcriptome maintenance.National Institutes of Health (U.S.) (grant GM035010)National Institutes of Health (U.S.) (grant GM040266

DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. Methodology/Principal Findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38uC and 42uC. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. Conclusion/Significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart

The Min System and Nucleoid Occlusion Are Not Required for Identifying the Division Site in Bacillus subtilis but Ensure Its Efficient Utilization

Precise temporal and spatial control of cell division is essential for progeny survival. The current general view is that precise positioning of the division site at midcell in rod-shaped bacteria is a result of the combined action of the Min system and nucleoid (chromosome) occlusion. Both systems prevent assembly of the cytokinetic Z ring at inappropriate places in the cell, restricting Z rings to the correct site at midcell. Here we show that in the bacterium Bacillus subtilis Z rings are positioned precisely at midcell in the complete absence of both these systems, revealing the existence of a mechanism independent of Min and nucleoid occlusion that identifies midcell in this organism. We further show that Z ring assembly at midcell is delayed in the absence of Min and Noc proteins, while at the same time FtsZ accumulates at other potential division sites. This suggests that a major role for Min and Noc is to ensure efficient utilization of the midcell division site by preventing Z ring assembly at potential division sites, including the cell poles. Our data lead us to propose a model in which spatial regulation of division in B. subtilis involves identification of the division site at midcell that requires Min and nucleoid occlusion to ensure efficient Z ring assembly there and only there, at the right time in the cell cycle

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ~2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.GM095733BBBE 103316MIT Startup Fun