Search

A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed. Author summary: Trypanosoma brucei is the causative agent of African trypanosomiasis (sleeping sickness) in humans and nagana in cattle. Its unusual genomic organisation featuring large polycistronic units requires a general mechanism of transcription initiation, because individual gene promoters are mostly absent. Despite the fact that the histone variant H2A.Z has previously been identified as a key player of transcription regulation, the complex responsible for correct H2A.Z incorporation at transcription start sites (TSS) remains elusive. In other eukaryotes, SWR1, a SNF2 ATPase-associated chromatin remodelling complex, is responsible for correct incorporation of this histone variant. This study identified a SWR1-like complex in T. brucei. Depletion of the SNF2 ATPase resulted in a reduction of H2A.Z incorporation at the TSS and decreased steady-state mRNA levels accompanied by chromatin condensation. In addition to the SWR1-like complex, we also identified a trypanosome-specific HAT2 complex that includes the histone acetyltransferases HAT2, a key player in the H2A.Z incorporation process. This complex has a trypanosome-specific composition that is different from the NuA4/TIP60 complex in Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

Chorioamnionitis is frequent in preterm labor and increases the risk of bronchopulmonary dysplasia. We hypothesized that intra-amniotic endotoxin injures the lung in utero, causing a sequence of inflammation and tissue injury similar to that which occurs in the injured adult lung. Preterm lamb lungs at 125 days gestational age were evaluated for indicators of inflammation, injury, and repair 5 h, 24 h, 72 h, and 7 days after 4 mg of intra-amniotic endotoxin injection. At 5 h, the epithelial cells in large airways expressed heat shock protein 70, and alveolar interleukin-8 was increased. Surfactant protein B (SP-B) decreased in alveolar type II cells at 5 h, and SP-B in lung tissue and alveolar lavage fluid increased by 72 h. By 24 h, neutrophils were recruited into the large airways, and cell death was the highest. Alveolar type II cells decreased by 25% at 24 h, and proliferation was highest at 72 h, consistent with tissue remodeling. Intraamniotic endotoxin caused surfactant secretion, inflammation, cell death, and remodeling as indications of lung injury. The recovery phase was accompanied by maturational changes in the fetal lung. surfactant; lung maturation; alveolar type II cells; respiratory distress syndrome; chorioamnionitis

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process. [ABSTRACT FROM AUTHOR]

Research was conducted to characterize sprouty, a key regulator of apical branching of the Drosophila airways and antagonist of fibroblast growth factor (FGF) signaling. Sibling control trains were analyzed with genomic fragments while developmental Northern blots were determined by phenol/chloro-form extraction of total RNA. Results indicated that the overactivity of the FGF homolog pathway led to a relaxation in the normal apical restriction to branching.

Abstract Background CCCH type zinc finger proteins are RNA binding proteins with regulatory functions at all stages of mRNA metabolism. The best-characterized member, tritetraproline (TTP), binds to AU rich elements in 3' UTRs of unstable mRNAs, mediating their degradation. In kinetoplastids, CCCH type zinc finger proteins have been identified as being involved in the regulation of the life cycle and possibly the cell cycle. To date, no systematic listing of CCCH proteins in kinetoplastids is available. Results We have identified the complete set of CCCH type zinc finger proteins in the available genomes of the kinetoplastid protozoa Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. One fifths (20%) of all CCCH motifs fall into non-conventional classes and many had not been previously identified. One third of all CCCH proteins have more than one CCCH motif, suggesting multivalent RNA binding. One third have additional recognizable domains. The vast majority are unique to Kinetoplastida or to a subgroup within. Two exceptions are of interest: the putative orthologue of the mRNA nuclear export factor Mex67 and a 3'-5' exoribonuclease restricted to Leishmania species. CCCH motifs are absent from these proteins in other organisms and might be unique, novel features of the Kinetoplastida homologues. Of the others, several have a predicted, and in one case experimentally confirmed, connection to the ubiquitination pathways, for instance a HECT-type E3 ubiquitin ligase. The total number of kinetoplastid CCCH proteins is similar to the number in higher eukaryotes but lower than in yeast. A comparison of the genomic loci between the Trypanosomatidae homologues provides insight into both the evolution of the CCCH proteins as well as the CCCH motifs. Conclusion This study provides the first systematic listing of the Kinetoplastida CCCH proteins. The number of CCCH proteins with more then one CCCH motif is larger than previously estimated, due to the identification of non-conventional CCCH motifs. Experimental approaches are now necessary to examine the functions of the many unique CCCH proteins as well as the function of the putative Mex67 and the Leishmania 3'-5' exoribonuclease.

Gradients of soil-nutrient distribution between trees and intercanopy areas are common in many semiarid woodland ecosystems. To test if microsites under and between canopies influenced P pool distribution in a semiarid woodland dominated by one-seed juniper [Juniperus monosperma (Engelm.) Sarg.] and galleta grass [Hilaria jamesii (Torr.) Benth.], we compared inorganic, organic, and microbial P pools under trees and intercanopy areas of two Aridisols. Soils collected (5-15 cm depth) under eight tree canopies and in eight intercanopy areas from a Calciorthid and a Camborthid were subjected to a sequential P fractionation scheme. Soils and microsites were significant independent factors determining total soil P, which ranged from 814 [micro]g P [g.sup.-1] soil (SE = 25) to 1123 [micro]g P [g.sup.-1] soil (SE = 21). Resin P was significantly influenced by the interaction of soils with microsite. Organic hydroxide P was the largest organic P fraction and exceeded or equaled the amount of resin P. It differed significantly between the Calciorthid at 10.1 [micro]g P [g.sup.-1] soil (SE = 1.0) and the Camborthid at 22.1 [micro]g P [g.sup.-1] soil (SE = 1.6). Microsite and soil did not significantly affect microbial P, which ranged from 12.9 [micro]g P [g.sup.-1] soil (SE = 2.1) to 17.0 [micro]g P [g.sup.-1] soil (SE = 0.7). Nutrients and microbial activity are usually concentrated under canopies in semiarid and arid ecosystems. This research shows that P pools distribution in the studied ecosystem did not follow this general pattern, and that soils may be more important in determining P pool distribution than microsites.

Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes. [ABSTRACT FROM AUTHOR]

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. [ABSTRACT FROM AUTHOR]

5’-3’ decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5’-3’ exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5’-3’ degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1) as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5’-3’ exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5’-3’ decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5’ polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA’s 5’ end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap. [ABSTRACT FROM AUTHOR]

Our primary objective was to follow the effects of altered C/N ratios of two residue materials on the abundance and potential activities of soil proteolytic bacteria during the initial stages of decomposition of organic residue derived nitrogen (N), taking into account the explicit physiological responses of spring wheat (SW; increase of C/N ratio) and faba bean (FB; decrease of C/N ratio) to elevated carbon dioxide (CO 2 ) exposure. FB and SW residues were obtained from a Mini-FACE experiment (ambient (380 μl l −1 ) CO 2 :C/N ratio FB: 100:1; SW: 75:1), elevated (550 μl l −1 ) CO 2 (FB: 42:1; SW: 105:1)) and incubated in a stagnic luvisol (Ap-horizon, 0–10 cm). Soil samples were obtained at 7, 14, 28, and 56 days of incubation and measured for potential activities of thermolysin-like neutral metalloproteases and alanyl-aminopeptidases as well as abundances of corresponding genes (i.e., neutral metalloproteases ( npr ) and alanine aminopeptidases ( pepN )). It was our major finding that distinct C/N ratios revealed only minor and inconsistent effects on gene abundances and potential enzyme activities. There was no relationship between activity potentials of both enzymes and abundances of corresponding genes implying the presence of inactive proteolytic bacteria. Moreover, a likely functional redundancy among proteolytic genes was noticed as explained by the omnipresence of Firmicutes (i.e., npr gene) as well as α- and β-proteobacteria (i.e., pepN gene) in studied soils. It was concluded that for prospective research, a greater variety of organic residue types from crops cultivated under elevated CO 2 with their individual biochemical characteristics should be considered to get a closer understanding of the ecological significance and complex dynamics of hitherto largely overlooked proteolytic bacteria in agricultural ecosystems. [ABSTRACT FROM AUTHOR]

For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival. [ABSTRACT FROM AUTHOR]

The flow of plant-derived carbon in soil is a key component of global carbon cycling. Conceptual models of trophic carbon fluxes in soil have assumed separate bacterial and fungal energy channels in the detritusphere, controlled by both substrate complexity and recalcitrance. However, detailed understanding of the key populations involved and niche-partitioning between them is limited. Here, a microcosm experiment was performed to trace the flow of detritusphere C from substrate analogs (glucose, cellulose) and plant biomass amendments (maize leaves, roots) in an agricultural soil. Carbon flow was traced by rRNA stable isotope probing and amplicon sequencing across three microbial kingdoms. Distinct lineages within the Actinobacteria, Bacteroidetes, Gammaproteobacteria, Basidiomycota, Ascomycota as well as Peronosporomycetes were identified as important primary substrate consumers. A dynamic succession of primary consumers was observed especially in the cellulose treatments, but also in plant amendments over time. While intra-kingdom niche partitioning was clearly observed, distinct bacterial and fungal energy channels were not apparent. Furthermore, while the diversity of primary substrate consumers did not notably increase with substrate complexity, consumer succession and secondary trophic links to bacterivorous and fungivorous microbes resulted in increased food web complexity in the more recalcitrant substrates. This suggests that rather than substrate-defined energy channels, consumer succession as well as intra- and inter-kingdom cross-feeding should be considered as mechanisms supporting food web complexity in the detritusphere. [ABSTRACT FROM AUTHOR]

The assemblages of organisms residing at different soil horizons are important drivers for carbon and nutrient cycle in arable systems, yet knowledge on food web dynamics below the plough zone is sparse. The present study investigated the effects of management practice on the nematode community structure and biomass in the top soil, rooted zone below plough layer, and root free deeper soil in two successive vegetation periods. Resource quality was manipulated by crop plant (maize and wheat) and organic amendment (with/without maize litter), to differently fuel the major soil carbon pathways based on roots, bacteria and fungi. Nematode assemblages were used as model to determine changes in the structure, function and carbon allocation of the soil micro-food web. The biomass of nematode families as well as the fungal to bacterial feeder ratio F/(F + B) assigned a predominance of the bacterial carbon channel in the arable soil. The differences in spatial and temporal availability of major plant resources, i.e. litter (recalcitrant substrate) and rhizodeposits (labile carbon), separated the nematode fauna into meta-communities along the depth profile. The seasonal pattern in population density, and the positive effects of litter amendment predominantly in the top soil, indicated low migration between these patches during crop growth. However, mass-flow as resource subsidy for communities at depth takes place under fallow during winter. Crop type was most important for carbon allocation, with wheat generally resulting in a greater biomass build-up of nematodes. The biomass distribution of trophic groups differed with depth, pointing to a strong bottom-up effect in the root channel, whereas predation likely was the primary regulating force in the fungal channel. Overall, combining nematode community composition with biomass revealed spatially separated micro-food web assemblages, differing in structure and carbon allocation. Thereby transport processes cross community boundaries, linking top soil and subsoil, notably in periods without a crop. [ABSTRACT FROM AUTHOR]

Plant-derived carbon (C) transfer to soil is one of the important factors controlling the size and structure of the belowground microbial community. The present study quantifies this plant-derived C incorporation into abiotic and biotic C pools in top- and subsoil in an arable field over five years. Stable isotope analysis was used to determine the incorporation of maize root and shoot litter C into soil organic C (SOC), extractable organic C (EOC), total microbial biomass (C mic ), ergosterol and phospholipid fatty acids (PLFAs). The following treatments were investigated: corn maize (CM), providing root- and shoot-derived C (without corncobs), fodder maize (FM), providing only root-derived C, and wheat plus maize shoot litter amendment (WL), providing only shoot-derived maize C. Wheat plants (W) without maize litter amendment served as control. Soil samples were taken each September directly before harvest from 2009 to 2013. During the experiment, the maize-derived C signal increased in SOC, EOC, C mic , ergosterol, bacterial and fungal PLFAs in the topsoil (0–10 cm). Although total maize shoot C input was threefold lower than maize root C input, similar relative amounts of maize C derived from shoots and roots were incorporated into the different C pools in the WL and the FM treatments, indicating the importance of shoot-derived C sources for microorganisms in the topsoil. An additive effect of both C sources was found in the CM treatment with almost twice as much maize-derived C in the respective pools. Furthermore, the proportion of maize-derived C varied between the different pools with lower incorporation into the total SOC (17%) and total EOC (24%) pools and higher incorporation ratios of maize C into PLFAs of different microbial groups (29% in Gram-positive (Gr + ) bacterial PLFA-C, 44% in Gram-negative (Gr − ) bacterial PLFA-C, 69% in fungal PLFA-C and 78% in ergosterol) in the CM treatment in topsoil after five years. After the third and fifth vegetation periods, we also detected maize-derived C in the rooted zone (40–50 cm depth) and the root-free zone (60–70 cm depth). The maize-derived C incorporation was lower in subsoil C pools in comparison to topsoil C pools. In the root-free zone, the maize-derived C was found to be 2% in total SOC, 28% in total EOC, 9% in Gr + bacterial PLFA-C, 20% in Gr − bacterial PLFA-C and 53% in fungal PLFA-C. Saprotrophic fungi incorporated maize-derived C in all soil depths to a greater degree than Gr + and Gr − bacteria, indicating the importance of saprotrophic fungi in this agro-ecosystem. [ABSTRACT FROM AUTHOR]

Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNAs from long polycistronic precursors by trans -splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5′-3′ exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNAs. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. [ABSTRACT FROM AUTHOR]

Background: The human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8-9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types. Results: A retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells. Conclusions: Our results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material. [ABSTRACT FROM AUTHOR]

Abstract: Many studies of the microbial ecology of agricultural ecosystems focus on surface soils, whereas the impacts of management practice and season on soil microbial community composition and function below the plough zone are largely neglected. Deep soils have a high potential to store carbon; therefore any management driven stimulation or repression of microorganisms in subsoil could impact biogeochemical cycling in agricultural sites. The aim of this study was to understand whether soil management affects microbial communities in the topsoil (0–10 cm), rooted zone beneath the plough layer (40–50 cm), and the unrooted zone (60–70 cm). In a field experiment with different crops [wheat (Triticum aestivum L.) and maize (Zea mays L.)] and agricultural management strategies (litter amendment) we analysed microbial biomass as phospholipid fatty acids (PLFAs) and enzyme activities involved in the C-cycle (β-glucosidase, N-acetyl-β-d-glucosaminidase, β-xylosidase, phenol- and peroxidase) across a depth transect over a period of two years. Wheat cultivation resulted in higher bacterial and fungal biomass as well as higher enzyme activities at most sampling dates in comparison to maize cultivated plots, and this effect was visible to 50 cm depth. Litter application increased bacterial and fungal biomass as well as hydrolytic enzyme activities but effects were apparent only in the topsoil. In winter high microbial biomass and enzyme activities were measured in all soil layers, possibly due to increased mobilization and translocation of organic matter into deeper soil. Hydrolytic enzyme activities decreased with depth, whereas oxidative enzyme activities showed no decrease or even an increase with depth. This could have been due to differing sorption mechanisms of hydrolytic and oxidative enzymes. Specific enzyme activities (enzyme activity per microbial biomass) were higher in the deeper layers and possible reasons are discussed. [Copyright &y& Elsevier]

The number of paralogs of proteins involved in translation initiation is larger in trypanosomes than in yeasts or many metazoan and includes two poly(A) binding proteins, PABP1 and PABP2, and four eIF4E variants. In many cases, the paralogs are individually essential and are thus unlikely to have redundant functions although, as yet, distinct functions of different isoforms have not been determined. Here, trypanosome PABP1 and PABP2 have been further characterised. PABP1 and PABP2 diverged subsequent to the differentiation of the Kinetoplastae lineage, supporting the existence of specific aspects of translation initiation regulation. PABP1 and PABP2 exhibit major differences in intracellular localization and distribution on polysome fractionation under various conditions that interfere with mRNA metabolism. Most striking are differences in localization to the four known types of inducible RNP granules. Moreover, only PABP2 but not PABP1 can accumulate in the nucleus. Taken together, these observations indicate that PABP1 and PABP2 likely associate with distinct populations of mRNAs. The differences in localization to inducible RNP granules also apply to paralogs of components of the eIF4F complex: eIF4E1 showed similar localization pattern to PABP2, whereas the localisation of eIF4E4 and eIF4G3 resembled that of PABP1. The grouping of translation initiation as either colocalizing with PABP1 or with PABP2 can be used to complement interaction studies to further define the translation initiation complexes in kinetoplastids. [ABSTRACT FROM AUTHOR]

Regulation of RNA polymerase II transcription initiation is apparently absent in trypanosomes. Instead, these eukaryotes control gene expression mainly at the post-transcriptional level. Regulation is exerted through the action of numerous RNAbinding proteins that modulate mRNA processing, turnover, translation and localization. In this work we show that the RNAbinding protein DRBD3 resides in the cytoplasm, but localizes to the nucleus upon oxidative challenge and to stress granules under starvation conditions. DRBD3 associates with other proteins to form a complex, the composition of which is altered by cellular stress. Interestingly, target mRNAs remain bound to DRBD3 under stress conditions. Our results suggest that DRBD3 transports regulated mRNAs within the cell in the form of ribonucleoprotein complexes that are remodeled in response to environmental cues. [ABSTRACT FROM AUTHOR]

Maturation of all cytoplasmic mRNAs in trypanosomes involves trans-splicing of a short exon at the 59 end. Inhibition of trans-splicing results in an accumulation of partially processed oligocistronic mRNAs. Here, we show that the accumulation of newly synthesised partially processed mRNAs results in the formation of foci around the periphery of the nucleus. These nuclear periphery granules (NPGs) contain the full complement of P-body proteins identified in trypanosomes to date, as well as poly(A)-binding protein 2 and the trypanosome homologue of the RNA helicase VASA. NPGs resemble perinuclear germ granules from metazoa more than P-bodies because they: (1) are localised around the nuclear periphery; (2) are dependent on active transcription; (3) are not dissipated by cycloheximide; (4) contain VASA; and (5) depend on nuclear integrity. In addition, NPGs can be induced in cells depleted of the P-body core component SCD6. The description of NPGs in trypanosomes provides evidence that there is a perinuclear compartment that can determine the fate of newly transcribed mRNAs and that germ granules could be a specialised derivative. [ABSTRACT FROM AUTHOR]

Abstract: The origin and quantity of plant inputs to soil are primary factors controlling the size and structure of the soil microbial community. The present study aimed to elucidate and quantify the carbon (C) flow from both root and shoot litter residues into soil organic, extractable, microbial and fungal C pools. Using the shift in C stable isotope values associated with replacing C3 by C4 plants we followed root- vs. shoot litter-derived C resources into different soil C pools. We established the following treatments: Corn Maize (CM), Fodder Maize (FM), Wheat+maize Litter (WL) and Wheat (W) as reference. The Corn Maize treatment provided root- as well as shoot litter-derived C (without corn cobs) whereas Fodder Maize (FM) provided only root-derived C (aboveground shoot material was removed). Maize shoot litter was applied on the Wheat+maize Litter (WL) plots to trace the incorporation of C4 litter C into soil microorganisms. Soil samples were taken three times per year (summer, autumn, winter) over two growing seasons. Maize-derived C signal was detectable after three to six months in the following pools: soil organic C (Corg), extractable organic C (EOC), microbial biomass (Cmic) and fungal biomass (ergosterol). In spite of the lower amounts of root- than of shoot litter-derived C inputs, similar amounts were incorporated into each of the C pools in the FM and WL treatments, indicating greater importance of the root- than shoot litter-derived resources for the soil microorganisms as a basis for the belowground food web. In the CM plots twice as much maize-derived C was incorporated into the pools. After two years, maize-derived C in the CM treatment contributed 14.1, 24.7, 46.6 and 76.2% to Corg, EOC, Cmic and ergosterol pools, respectively. Fungi incorporated maize-derived C to a greater extent than did total soil microbial biomass. [Copyright &y& Elsevier]

Abstract: Kinetoplastids, including the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania, are the only known organisms that do not regulate the transcription of protein coding genes transcribed by RNA polymerase II. Yet, profound changes in gene expression are induced by many different external stimuli and stresses, the extreme example are cascades of changes in gene expression initiated by differentiation triggers that ultimately and irreversibly result in the massive morphological and metabolic changes observed during life-cycle progression. This review explores how kinetoplastids change gene expression by looking at life-cycle stage specific changes in chromatin, mRNA processing, mRNA stability, mRNA translation, protein stability and protein modifications. [Copyright &y& Elsevier]

Background: CCCH type zinc finger proteins are RNA binding proteins with regulatory functions at all stages of mRNA metabolism. The best-characterized member, tritetraproline (TTP), binds to AU rich elements in 3' UTRs of unstable mRNAs, mediating their degradation. In kinetoplastids, CCCH type zinc finger proteins have been identified as being involved in the regulation of the life cycle and possibly the cell cycle. To date, no systematic listing of CCCH proteins in kinetoplastids is available. Results: We have identified the complete set of CCCH type zinc finger proteins in the available genomes of the kinetoplastid protozoa Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. One fifths (20%) of all CCCH motifs fall into non-conventional classes and many had not been previously identified. One third of all CCCH proteins have more than one CCCH motif, suggesting multivalent RNA binding. One third have additional recognizable domains. The vast majority are unique to Kinetoplastida or to a subgroup within. Two exceptions are of interest: the putative orthologue of the mRNA nuclear export factor Mex67 and a 3'-5' exoribonuclease restricted to Leishmania species. CCCH motifs are absent from these proteins in other organisms and might be unique, novel features of the Kinetoplastida homologues. Of the others, several have a predicted, and in one case experimentally confirmed, connection to the ubiquitination pathways, for instance a HECT-type E3 ubiquitin ligase. The total number of kinetoplastid CCCH proteins is similar to the number in higher eukaryotes but lower than in yeast. A comparison of the genomic loci between the Trypanosomatidae homologues provides insight into both the evolution of the CCCH proteins as well as the CCCH motifs. Conclusion: This study provides the first systematic listing of the Kinetoplastida CCCH proteins. The number of CCCH proteins with more then one CCCH motif is larger than previously estimated, due to the identification of nonconventional CCCH motifs. Experimental approaches are now necessary to examine the functions of the many unique CCCH proteins as well as the function of the putative Mex67 and the Leishmania 3'-5' exoribonuclease. [ABSTRACT FROM AUTHOR]

Background: The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest. Methodology/Principal Findings: Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1-4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells. Conclusions: The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint. [ABSTRACT FROM AUTHOR]

Abstract: When conservation tillage is practised in agriculture, plant residues remain on the soil surface for soil protection purposes. These residues should be widely decomposed within the following vegetation period as microbial plant pathogens surviving on plant litter may endanger the currently cultivated crop. Important soil-borne fungal pathogens that preferably infect small grain cereals belong to the genus Fusarium. These pathogens produce the mycotoxin deoxynivalenol (DON), a cytotoxic agent, in infected cereal organs. This toxin frequently occurs in cereal residues like straw. So far it is unclear if DON degradation is affected by members of the soil food web within decomposing processes in the soil system. For this purpose, a microcosm study was conducted under controlled laboratory conditions to investigate the degradation activity of the earthworm species Lumbricus terrestris when exposed to Fusarium-infected wheat straw being contaminated with DON. Highly Fusarium-infected and DON-contaminated straw seemed to be more attractive to L. terrestris because it was incorporated faster into the soil compared with straw infected and contaminated at low levels. This is supported by a greater body weight gain (exposure time 5 weeks) and smaller body weight loss (exposure time 11 weeks) of L. terrestris, respectively, when highly contaminated straw was offered for different time periods. Furthermore, L. terrestris takes part in the efficient degradation of both Fusarium biomass and DON occurring in straw in close interaction with soil microorganisms. Consequently, earthworm activity contributes to the elimination of potentially infectious plant material from the soil surface. [Copyright &y& Elsevier]

The integration of closed biomass cycles into residential buildings enables efficient resource utilization and avoids the transport of biowaste. In our scenario called Integrated Cycles for Urban Biomass (ICU), biowaste is degraded on-site into biogas that is converted into heat and electricity. Nitrification processes upgrade the liquid fermentation residues to refined fertilizer, which can be used subsequently in house-internal gardens to produce fresh food for residents. Our research aims to assess the ICU scenario regarding produced amounts of biogas and food, saved CO2 emissions and costs, and social–cultural aspects. Therefore, a model-based feasibility study was performed assuming a building with 100 residents. The calculations show that the ICU concept produces 21% of the annual power (electrical and heat) consumption from the accumulated biowaste and up to 7.6 t of the fresh mass of lettuce per year in a 70 m2 professional hydroponic production area. Furthermore, it saves 6468 kg CO2-equivalent (CO2-eq) per year. While the ICU concept is technically feasible, it becomes economically feasible for large-scale implementations and higher food prices. Overall, this study demonstrates that the ICU implementation can be a worthwhile contribution towards a sustainable CO2-neutral society and decrease the demand for agricultural land. [ABSTRACT FROM AUTHOR]

Most insertional mutagenesis screens of Drosophila performed to date have not used target chromosomes that have been checked for their suitability for phenotypic screens for viable phenotypes. To address this, we have generated a selection of stocks carrying either isogenized second chromosomes or isogenized third chromosomes, in a genetic background derived from a Canton-S wild-type strain. We have tested these stocks for a range of behavioral and other viable phenotypes. As expected, most lines are statistically indistinguishable from Canton-S in most phenotypes tested. The lines generated are now being used as target chromosomes in mutagenesis screens, and the characterization reported here will facilitate their use in screens of these lines for behavioral and other viable phenotypes. [ABSTRACT FROM AUTHOR]

Investigates the inorganic, organic and microbial phosphorus pools under trees and intercanopy areas of two ardisols. Soils and microsites as significant independent factors in determining total soil phosphorus; Concentration of nutrients and microbial activity under canopies in arid and semiarid ecosystems.