Microbial community succession during lactate amendment and electron acceptor limitation reveals a predominance of metal-reducing Pelosinus spp

The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N(2) gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.     

Assessing the Viral Fitness of Oseltamivir-Resistant Influenza Viruses in Ferrets,Using a Competitive-Mixtures Model

To determine the relative fitness of oseltamivir-resistant strains compared to susceptible wild-type viruses, we combined mathematical modeling and statistical techniques with a novel in vivo “competitive-mixtures” experimental model. Ferrets were coinfected with either pure populations (100% susceptible wild-type or 100% oseltamivir-resistant mutant virus) or mixed populations of wild-type and oseltamivir-resistant influenza viruses (80%:20%, 50%:50%, and 20%:80%) at equivalent infectivity titers, and the changes in the relative proportions of those two viruses were monitored over the course of the infection during within-host and over host-to-host transmission events in a ferret contact model. Coinfection of ferrets with mixtures of an oseltamivir-resistant R292K mutant A(H3N2) virus and a R292 oseltamivir-susceptible wild-type virus demonstrated that the R292K mutant virus was rapidly outgrown by the R292 wild-type virus in artificially infected donor ferrets and did not transmit to any of the recipient ferrets. The competitive-mixtures model was also used to investigate the fitness of the seasonal A(H1N1) oseltamivir-resistant H274Y mutant and showed that within infected ferrets the H274Y mutant virus was marginally outgrown by the wild-type strain but demonstrated equivalent transmissibility between ferrets. This novel in vivo experimental method and accompanying mathematical analysis provide greater insight into the relative fitness, both within the host and between hosts, of two different influenza virus strains compared to more traditional methods that infect ferrets with only pure populations of viruses. Our statistical inferences are essential for the development of the next generation of mathematical models of the emergence and spread of oseltamivir-resistant influenza in human populations.The neuraminidase (NA) inhibitors are a class of influenza antiviral drugs that are specifically designed to inhibit the enzymatic function of the NA, thereby preventing normal viral replication. Since 1999, two NA inhibitors (NAIs), oseltamivir (Tamiflu) and zanamivir (Relenza), have been shown to be effective for the treatment and prophylaxis of patients infected with not only seasonal influenza, but also highly pathogenic A(H5N1) and the newly emerged A(H1N1) pandemic virus. Prior to 2007, resistance to this class of drugs was considered relatively uncommon, particularly in comparison with the other class of influenza antivirals, the adamantanes, which readily select for viral resistance in treated patients. During early clinical trials, oseltamivir resistance was detected in only 1 to 2% of adults (14) and 5 to 6% of children (33) under treatment, although later studies detected resistance in up to 18% of oseltamivir-treated children (16). In contrast, resistance following zanamivir treatment is rare, with only one reported case observed in an immunocompromised patient (6). Influenza viruses that develop resistance to these drugs typically contain mutations in the NA which, either directly or indirectly, alter the shape of the NA enzymatic site, thereby reducing the ability of the drugs to bind to this specific pocket. One of the most commonly observed mutations in oseltamivir-resistant A(H3N2) viruses is an arginine-to-lysine mutation at residue 292 (R292K) of the NA, while the predominant NA mutation in oseltamivir-resistant A(H1N1) viruses is a histidine-to-tyrosine mutation at residue 274 (H274Y) (N2 NA amino acid numbering, equivalent to residue 275 based on N1 numbering). Both of these mutations have an indirect impact on drug binding, as they affect the ability of the glutamic acid residue at position 276 to reorientate, as required for slow binding by oseltamivir (3). Many mutations that cause NAI resistance also cause reduced NA enzyme activity and, consequently, can compromise viral fitness.Previous studies have demonstrated that viruses with an R292K NA mutation demonstrated compromised growth in vitro (36) and in ferrets were significantly less infectious and did not transmit (9). The replication and transmission fitness of the H274Y mutation has also been studied previously. An H274Y mutant A(H1N1) strain isolated from a patient under oseltamivir treatment demonstrated compromised growth in cell culture compared to a wild-type (WT) virus (13), although a strain carrying the same mutation selected in vitro was found to replicate as well as the wild type (32). The infectivity and transmissibility of an H274Y mutant were found to be restricted in ferrets (13), although a second study demonstrated that transmission of the mutant virus between ferrets was possible, but required a greater viral dose of the mutant compared to the wild type (10). These results suggest that resistant virus variants with the same NA mutation may differ in replication or transmission fitness depending on other viral components. Nevertheless, based on these data and the viral fitness of other resistant mutants, it was believed that NAI-resistant viruses were unlikely to spread throughout the community due to their compromised viral fitness in the absence of drug selective pressure. This was proven incorrect during the Northern Hemisphere 2007-2008 influenza season, when large numbers of oseltamivir-resistant seasonal A(H1N1) viruses with an H274Y mutation were detected in patients who had not been treated with oseltamivir (4, 24). The mutant strain continued to spread to the Southern Hemisphere, such that by late 2008 virtually all circulating seasonal A(H1N1) viruses were oseltamivir resistant (11). The rapid global spread of this strain clearly suggested that the oseltamivir-resistant seasonal A(H1N1) virus had fitness equivalent to or greater than that of the previous oseltamivir-sensitive A(H1N1) strain. The reasons for enhanced viral fitness in this strain, when previous studies demonstrated that the acquisition of an H274Y mutation led to reduced viral fitness, remain unclear but probably involve compensatory mutations or reassortment events which may have improved the hemagglutinin (HA)/NA balance, allowing efficient transmission (5, 26).Experimental methods have been developed to assess the relative fitness of NAI-resistant strains compared with respective wild-type viruses, both in vitro and in vivo. Ferrets have been considered the most appropriate model animal for influenza research, and fitness studies have assessed variables such as minimum dose required to achieve infection, duration of viral shedding, and levels of viral load to allow comparisons between viruses. The guinea pig model has also been previously used to assess the viral fitness of influenza viruses, particularly in comparing the transmissibility of strains via either the contact or aerosol route (2). As an alternative to these traditional approaches, we have investigated a methodology that involves coinfection of ferrets with a mixture of two influenza viruses. Daily monitoring of changes in the relative proportion of those viruses over the course of the infection allows determination of the relative replication fitness of the viruses. Monitoring of recipient ferrets exposed to the infected ferrets enables the relative transmissibility of the viruses (henceforth, the relative transmission fitness) to be determined. In this study, the “competitive-mixtures” methodology was used to assess the relative replication and transmission fitness of an oseltamivir-resistant R292K mutant A(H3N2) virus compared with an oseltamivir-sensitive A(H3N2) wild-type strain and also to asses the relative replication and transmission fitness of an oseltamivir-resistant H274Y seasonal A(H1N1) mutant compared with an oseltamivir-sensitive A(H1N1) wild-type strain. Quantitative estimates for the replication fitness of mutant viruses were determined using a simple mathematical model of within-host viral replication and mixed-effects statistical tests. Transmission fitness was evaluated by application of a graphical technique that demonstrated the relationship between the proportion of mutant virus in the infectee ferrets as a function of the proportion of mutant virus in the infector ferrets.Inferences drawn from the statistical analyses presented here are essential for the refinement of existing mathematical models that simulate the spread of influenza in the human population and model the deployment of antiviral agents. These models are designed to assess the likely impact of different antiviral agent deployment strategies to control pandemic influenza (18, 21, 35). At present, data on the probability of emergence of NAI-resistant strains, the relative transmission fitness of these strains, and the probability of an individual''s infection reverting to an NAI-sensitive strain in the absence of ongoing selective pressure are severely limited. In consequence, human population-level models of influenza spread must make gross assumptions on the likely characteristics of NAI-resistant strains. Data such as those presented here will be used to inform new models of drug deployment and result in improved pandemic policy advice (20, 23).     

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

Background  

The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor.     

Formation and nuclear export of preribosomes are functionally linked to the small-ubiquitin-related modifier pathway

Ribosomal precursor particles are initially assembled in the nucleolus prior to their transfer to the nucleoplasm and export to the cytoplasm. In a screen to identify thermosensitive (ts) mutants defective in the export of pre-60S ribosomal subunit, we isolated the rix16-1 mutant. In this strain, nucleolar accumulation of the Rpl25-eGFP reporter was complemented by UBA2 (a subunit of the E1 sumoylation enzyme). Mutations in UBC9 (E2 enzyme), ULP1 [small-ubiquitin-related modifier (SUMO) isopeptidase] and SMT3 (SUMO-1) caused 60S export defects. A directed analysis of the SUMO proteome revealed that many ribosome biogenesis factors are sumoylated. Importantly, preribosomal particles along both the 60S and the 40S synthesis pathways were decorated with SUMO, showing its direct involvement. Consistent with this, early 60S assembly factors were genetically linked to SUMO conjugation. Notably, the SUMO deconjugating enzyme Ulp1, which localizes to the nuclear pore complex (NPC), was functionally linked to the 60S export factor Mtr2. Together our data suggest that sumoylation of preribosomal particles in the nucleus and subsequent desumoylation at the NPC is necessary for efficient ribosome biogenesis and export in eukaryotes.     

Coordinated nuclear import of RNA polymerase III subunits

Eukaryotic RNA polymerases are multisubunit assemblies, whose enzymatic function in the nucleus is intensively studied. However, little is known about the biogenesis of the three RNA polymerases and coupling to nucleo-cytoplasmic transport. Here, we show that Rpc128, the second largest subunit of RNA polymerase III, was mislocalized to the cytoplasm, when a short sequence in the N-terminal domain was deleted. Importantly, nuclear import of other, but not all, RNA polymerase III subunits was impaired in this RPC128DeltaN mutant. These data suggest that RNA polymerase III subunits are not imported independently into the nucleus but may require preassembly into cytoplasmic subcomplexes for coordinated nuclear uptake. We expect these studies to be a starting point to dissect the complex biogenesis pathway of eukaryotic RNA polymerases.     

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.     

Initial stages of reproductive isolation in two species of the endangered Sonoran topminnow

Long-term geographic isolation can result in reproductive incompatibilities due to forces such as mutation, genetic drift, and differential selection. In the Sonoran topminnow, molecular genetic studies of mtDNA, microsatellites, and MHC genes have shown that the endangered Gila and Yaqui topminnows are substantially different, suggesting that divergence took place approximately two million years ago. Here we examined hybrid crosses and backcrosses between these two allopatric taxa to evaluate the accumulation of postmating barriers to reproduction. These results are then compared with results from a previous study where male topminnows were shown to mate assortatively with conspecific females. Despite their preference for conspecific mates, both types of interspecific crosses successfully produced offspring. There was evidence of reduced hybrid fitness, including smaller mean brood size and male-biased sex ratio, for some classes of backcrosses. Brood sizes and interbrood intervals varied significantly when hybrids were subdivided into different cross categories. Our results illustrate the importance of distinctly defining hybrid classes in studies of reproductive isolation. To our knowledge, this is the first such detailed evolutionary analysis in endangered fish taxa.     

Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.     

Structure of the C-terminal FG-nucleoporin binding domain of Tap/NXF1

The vertebrate Tap protein is a member of the NXF family of shuttling transport receptors for nuclear export of mRNA. Tap has a modular structure, and its most C-terminal domain is important for binding to FG repeat-containing nuclear pore proteins (FG-nucleoporins) and is sufficient to mediate nuclear shuttling. We report the solution structure of this C-terminal domain, which is based on a distinctive arrangement of four alpha-helices and is joined to the next module by a flexible 12-residue Pro-rich linker. F617A Tap suppresses FG-nucleoporin binding by the most C-terminal domain that, together with the structure of the other modules from which Tap is constructed, provides a structural context for its nuclear shuttling function.