Search

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

The passage of mRNAs through the nuclear pores into the cytoplasm is essential in all eukaryotes. For regulation, mRNA export is tightly connected to the full machinery of nuclear mRNA processing, starting at transcription. Export competence of pre-mRNAs gradually increases by both transient and permanent interactions with multiple RNA processing and export factors. mRNA export is best understood in opisthokonts, with limited knowledge in plants and protozoa. Here, I review and compare nuclear mRNA processing and export between opisthokonts and Trypanosoma brucei. The parasite has many unusual features in nuclear mRNA processing, such as polycistronic transcription and trans-splicing. It lacks several nuclear complexes and nuclear-pore-associated proteins that in opisthokonts play major roles in mRNA export. As a consequence, trypanosome mRNA export control is not tight and export can even start co-transcriptionally. Whether trypanosomes regulate mRNA export at all, or whether leakage of immature mRNA to the cytoplasm is kept to a low level by a fast kinetics of mRNA processing remains to be investigated. mRNA export had to be present in the last common ancestor of eukaryotes. Trypanosomes are evolutionary very distant from opisthokonts and a comparison helps understanding the evolution of mRNA export.

Mean sensitivity (MS) derived from a standard test grid using microperimetry is a sensitive outcome measure in clinical trials investigating new treatments for degenerative retinal diseases. This study hypothesizes that the functional decline is faster at the edge of the dense scotoma (eMS) than by using the overall MS.Multicenter, international, prospective cohort study: ProgStar Study.Stargardt disease type 1 patients (carrying at least 1 mutation in the ABCA4 gene) were followed over 12 months using microperimetry with a Humphrey 10-2 test grid. Customized software was developed to automatically define and selectively follow the test points directly adjacent to the dense scotoma points and to calculate their mean sensitivity (eMS).Among 361 eyes (185 patients), the mean age was 32.9 ± 15.1 years old. At baseline, MS was 10.4 ± 5.2 dB (n = 361), and the eMS was 9.3 ± 3.3 dB (n = 335). The yearly progression rate of MS (1.5 ± 2.1 dB/year) was significantly lower (β = -1.33; P.001) than that for eMS (2.9 ± 2.9 dB/year). There were no differences between progression rates using automated grading and those using manual grading (β = .09; P = .461).In Stargardt disease type 1, macular sensitivity declines significantly faster at the edge of the dense scotoma than in the overall test grid. An automated, time-efficient approach for extracting and grading eMS is possible and appears valid. Thus, eMS offers a valuable tool and sensitive outcome parameter with which to follow Stargardt patients in clinical trials, allowing clinical trial designs with shorter duration and/or smaller cohorts.

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.

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.

Numerous 13CO2 labeling studies have traced the flow of carbon from fresh plant exudates into rhizosphere bacterial communities. However, the succession of the uptake of carbon leaving the roots by distinct rhizosphere microbiota has rarely been resolved between microbial kingdoms. This can provide valuable insights on the niche partitioning of primary rhizodeposit consumption, as well as on community interactions in plant-derived carbon flows in soil. Here, we have traced the flow of fresh plant assimilates to rhizosphere microbiota of maize (Zea mays L.) by rRNA-stable isotope probing (SIP). Carbon flows involving bacteria, unicellular fungi, as well as protists were observed over 5 and 8 days. Surprisingly, labeling of Paraglomerales and several bacteria including Opitutus, Mucliaginibacter and Massilia spp. was especially apparent in soil surrounding the strict rhizosphere after 5 d. This highlights the central role of arbuscular mycorrhizal fungi (AMF) as a shunt for fresh plant assimilates to soil microbes not directly influenced by root exudation. Distinct trophic webs involving different flagellates, amoeba and ciliates were also observed in rhizosphere and surrounding soil, while labeling of filamentous saprotrophic Ascomycota or Basidiomycota was not apparent. This challenges the proposed “sapro-rhizosphere” concept and demonstrates the utility of rRNA-SIP to disentangle inter-kingdom microbial relationships in the rhizosphere.

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC50 ≥ 6.5 nM) and high affinity ligands (KD ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens., Protein kinase A (PKA) is typically activated by cAMP. Here, Bachmaier et al. show that PKA of Trypanosoma is activated by nucleoside-related ligands, explain the ligand selectivity swap by a co-crystal structure of trypanosome PKAR, and identify potential downstream targets by phosphoproteomics.

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 summaryTrypanosoma 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.

BackgroundApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and are present in eukaryotes of all eukaryotic super-groups; still, only two proteins have been functionally characterised. One is ALPH1 from the KinetoplastidTrypanosoma bruceithat we recently found to be the mRNA decapping enzyme of the parasite. mRNA decapping by ALPHs is unprecedented in eukaryotes, which usually use nudix hydrolases, but the bacterial ancestor protein ApaH was recently found to decap non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or more widespread among eukaryotes.ResultsWe screened 824 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to refine phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of ALPHs. We found that most eukaryotes have either no ALPH (500/824) or very short ALPHs, consisting almost exclusively of the catalytic domain. These ALPHs had mostly predicted non-cytoplasmic localisations, often supported by the presence of transmembrane helices and signal peptides and in two cases (one in this study) by experimental data. The only exceptions were ALPH1 homologues from Kinetoplastida, that all have unique C-terminal and mostly unique N-terminal extension, and at least theT. bruceienzyme localises to the cytoplasm. Surprisingly, despite of these non-cytoplasmic localisations, ALPHs from all eukaryotic super-groups hadin vitromRNA decapping activity.ConclusionsALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme since, or use it exclusively outside the cytoplasm in organelles in a version consisting of the catalytic domain only. While our data provide no evidence for the presence of further mRNA decapping enzymes among eukaryotic ALPHs, the broad substrate range of ALPHs that includes mRNA caps provides an explanation for the selection against the presence of a cytoplasmic ALPH protein as a mean to protect mRNAs from unregulated degradation. Kinetoplastida succeeded to exploit ALPH as their mRNA decapping enzyme, likely using the Kinetoplastida-unique N- and C-terminal extensions for regulation.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridisation (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs.To overcome these limitations, we have hybridised slices of high pressure frozen, LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localisation that can be complemented with immunofluorescence and electron microscopy, as well as electron tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA tertiary structures, and we show that the method can be employed to probe for mRNA compactness. We apply LR White smFISH to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing, using trypanosomes and their versatile RNA biology as a model system.

Purpose To investigate the natural history of Stargardt disease (STGD1) using fixation location and fixation stability. Design Multicenter, international, prospective cohort study. Methods Fixation testing was performed using the Nidek MP-1 microperimeter as part of the prospective, multicenter, natural history study on the Prog ression of Star gardt disease (ProgStar). A total of 238 patients with ABCA4-related STGD1 were enrolled at baseline (bilateral enrollment in 86.6%) and underwent repeat testing at months 6 and 12. Results Outcome measures included the distance of the preferred retinal locus from the fovea (PRL) and the bivariate contour ellipse area (BCEA). After 12 months of follow-up, the change in the eccentricity of the PRL from the anatomic fovea was −0.0014 degrees (95% confidence interval [CI], −0.27 degrees, 0.27 degrees; P = .99). The deterioration in the stability of fixation as expressed by a larger BCEA encompassing 1 standard deviation of all fixation points was 1.21 degrees squared (deg2) (95% CI, −1.23 deg2, 3.65 deg2; P = .33). Eyes with increases and decreases in PRL eccentricity and/or BCEA values were observed. Conclusions Our observations point to the complexity of fixation parameters. The association of increasingly eccentric and unstable fixation with longer disease duration that is typically found in cross-sectional studies may be countered within individual patients by poorly understood processes like neuronal adaptation. Nevertheless, fixation parameters may serve as useful secondary outcome parameters in selected cases and for counseling patients to explain changes to their visual functionality.

This study assessed the carbon (C) budget and the C stocks in major compartments of the soil food web (bacteria, fungi, protists, nematodes, meso- and macrofauna) in an arable field with/without litter addition. The C stocks in the food web were more than three times higher in topsoil (0–10 cm) compared to subsoil (>40 cm). Microorganisms contained over 95% of food web C, with similar contributions of bacteria and fungi in topsoil. Litter addition did not alter C pools of soil biota after one growing season, except for the increase of fungi and fungal feeding nematodes in the topsoil. However, the C budget for functional groups changed with depth, particularly in the microfauna. This suggests food web resilience to litter amendment in terms of C pool sizes after one growing season. In contrast, the distinct depth dependent pattern indicates specific metacommunities, likely shaped by dominant abiotic and biotic habitat properties.

Decomposition of organic matter is crucial for ecosystem functioning. Microorganisms, which are responsible for the mineralization of organic matter, are usually treated as a homogeneous functional guild, despite mineralization capacity can differ profoundly between taxa. In addition, a significant part of the microbial community is top-down controlled by microbial grazers, such as protist. Since protist grazing is selective, and selectivity differs among species, we hypothesized that protist taxa complement each other in grazing intensity and thereby affect bacterial community structure and mineralization rate. In a laboratory experiment the species richness of protist communities was manipulated in an arable field soil and the mineralization rate of maize litter residues followed during the decomposition of the labile (4 days) and recalcitrant (3 weeks) carbon (C) fractions. Mineralization rate overall increased in the presence of protists. Changes in microbial function could be related to changes in microbial community composition (measured by phospholipid fatty acids pattern). During microbial decomposition, different protist grazers gained influence on mineralization rates over consecutive time intervals, indicating that a succession of protists caused an enhanced bacterial C-mineralization of plant detritus. Protist identity and species richness affected the microbial community composition, but not the magnitude of its mineralization function. In general, protist identity appeared to be more relevant for the composition of the microbial communities at the beginning of decomposition while the protist species richness appeared to be more critical in the later, slow phase of decomposition. This study provides an example that the overall outcome of ecosystem processes, such as mineralization rate is regulated by the sum of positive and negative effects of complex species interactions operating at a very fine spatial and temporal scales.

Agroecosystems occupy large areas of the land surface and arable soil food webs are of significant importance for global cycling of carbon (C) and nitrogen (N). In a field experiment we labeled maize plants ( Zea mays L.) in 13 CO 2 atmosphere and by K 15 NO 3 fertilization. During 25 days, the incorporation of 13 C and 15 N was traced in plant compartments, soil and soil arthropods, as well as 13 C in microbial phospholipid fatty acids (PLFAs) and nematodes. Highlighting the importance of root-derived resources in agroecosystems, 13 C was incorporated into all food web compartments, including microorganisms (PLFAs), nematodes and arthropods. The amount of incorporated 13 C (as compared to unlabeled samples) markedly decreased along the food chain with ∆ 13 C decreasing from 500‰ in plant roots and 900‰ in microbial PLFAs, to less than 40‰ in nematodes and arthropods. Incorporation of 13 C into fungal PLFAs considerably exceeded that into bacterial PLFAs, highlighting the importance of soil fungi as compared to bacteria in C cycling. Fertilizer-derived 15 N uniformly increased with time in plant compartments and soil arthropods, indicating that N is distributed homogeneously in the soil food web. High channeling of both root-derived 13 C and fertilizer-derived 15 N to higher trophic levels by fungi, and intensive feeding on fungi by soil animals highlight the central role of saprotrophic fungi in C and nutrient fluxes in soil food webs of arable ecosystems.

The 5' ends of most RNAs are chemically modified to enable protection from nucleases. In bacteria, this is often achieved by keeping the triphosphate terminus originating from transcriptional initiation, while most eukaryotic mRNAs and small nuclear RNAs have a 5'→5' linked N7 -methyl guanosine (m7 G) cap added. Several other chemical modifications have been described at RNA 5' ends. Common to all modifications is the presence of at least one pyrophosphate bond. To enable RNA turnover, these chemical modifications at the RNA 5' end need to be reversible. Dependent on the direction of the RNA decay pathway (5'→3' or 3'→5'), some enzymes cleave the 5'→5' cap linkage of intact RNAs to initiate decay, while others act as scavengers and hydrolyse the cap element of the remnants of the 3'→5' decay pathway. In eukaryotes, there is also a cap quality control pathway. Most enzymes involved in the cleavage of the RNA 5' ends are pyrophosphohydrolases, with only a few having (additional) 5' triphosphonucleotide hydrolase activities. Despite the identity of their enzyme activities, the enzymes belong to four different enzyme classes. Nudix hydrolases decap intact RNAs as part of the 5'→3' decay pathway, DXO family members mainly degrade faulty RNAs, members of the histidine triad (HIT) family are scavenger proteins, while an ApaH-like phosphatase is the major mRNA decay enzyme of trypanosomes, whose RNAs have a unique cap structure. Many novel cap structures and decapping enzymes have only recently been discovered, indicating that we are only beginning to understand the mechanisms of RNA decapping. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA Processing > Capping and 5' End Modifications.

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.

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 (Cmic), 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, Cmic, 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.

Mycorrhizal fungi enhance plant phosphorus (P) acquisition via their extraradical hyphae (ERH) that scavenge nutrients outside root depletion zones. While soil P availability declines during ecosystem retrogression, how ERH biomass and scavenging vary during ecosystem retrogression remains unknown; it is expected to increase if plants allocate more carbon (C) to mycorrhizal fungi as P availability declines. We measured fungal and bacterial biomass using in-growth cores and lipid biomarkers along a 2-million-year dune chronosequence in an Australian biodiversity hotspot showing a ~60-fold decline in total soil P concentration with increasing soil age. We compared the levels of key fungal biomarkers (ergosterol, NLFA 16:1ω5, and PLFA 18:2ω6,9) between closed, mesh, and open cores during five months (four sampling dates including the wet winter months), thus allowing us to also determine the dynamics of mycorrhizal fungal scavenging.We found strikingly low and declining biomass of ERH with declining P availability, with minimal long-distance scavenging by ERH. Biomass of ERH was highest in the younger (c. 1 ka) soils that were comparatively rich in P and other nutrients. By contrast, the oldest, most P impoverished soils had the lowest biomass of ERH, despite high mycorrhizal root colonisation, and high abundance and diversity of potential plant hosts. We show that extremely low P availability constrains ERH biomass. Such low mycorrhizal fungal biomass highlights the need for a more 'mycocentric' view of plant-mycorrhizal relationships in old, severely P-impoverished ecosystems. Fil: Teste, Francois. University of Western Australia; Australia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina Fil: Laliberté, Etienne. University of Western Australia; Australia. University of Montreal; Canadá Fil: Lambers, Hans. University of Western Australia; Australia Fil: Auer, Yasha. University Of Hohenheim; Alemania Fil: Kramer, Susanne. University Of Hohenheim; Alemania Fil: Kandeler, Ellen. University Of Hohenheim; Alemania

The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.

Summary 1. The complexity of soil food webs and the cryptic habitat hamper the analyses of pools, fluxes and turnover rates of carbon (C) in organisms and the insight into their interactions. Stable isotope analysis has been increasingly used to disentangle soil food web structure, yet it has not been applied to quantitatively characterize C dynamics at the level of the entire soil food web. 2. The present study employed 13 CO2 pulse labelling to investigate the incorporation of maize root-derived C into major soil compartments and food web players in an arable field for 25 days. Bulk tissue and compound-specific (lipids) C isotope ratios were used to quantify pool sizes and 13 C incorporation in bacteria and fungi as primary decomposers, nematodes as key drivers of the microfood web and decomposers and predators among the meso- and macrofauna. 3. About 20% of the C assimilated by maize was transferred to below-ground pools. 13 C was predominantly incorporated into rhizosphere micro-organisms rather than in those of the bulk soil. 13 C in phospholipid fatty acid biomarkers revealed that root-derived C was incorporated into the soil food web mainly via saprotrophic fungi rather than via bacteria. Only small amounts of 13 C were transferred to higher trophic levels, predominantly into fungal-feeding nematodes and macrofauna decomposers. 4. Most importantly, C pool size and 13 C incorporation did not match closely. Although the fungal C stock was less than half that of bacteria, C transfers from fungi into higher trophic levels of the fungal energy pathway, that is fungal-feeding nematodes and meso- and macrofauna decomposers, by far exceed that of bacterial C. This challenges previous views on the dominance of bacteria in root C dynamics and suggests saprotrophic fungi to function as major agents channelling recent photoassimilates into the soil food web.

Soil fungi play an essential role in the decomposition of plant-derived organic material entering soils. The quality and quantity of organic compounds vary seasonally as well as with soil depth. To elucidate how these resources affect fungal communities in an arable soil, a field experiment was set up with two plant species, maize and wheat. Resource availability was experimentally manipulated by maize litter input on one half of these maize and wheat plots after harvest in autumn. Fungal biomass was determined by ergosterol quantification, and community structure was investigated by fungal automated ribosomal intergenic spacer analysis (F-ARISA). An annual cycle was assessed across a depth gradient, distinguishing three soil habitats: the plough layer, rooted soil below the plough layer, and deeper root-free soil. Fungal communities appeared highly dynamic and varied according to soil depth and plant resources. In the plough layer, the availability of litter played a dominant role in shaping fungal communities, whereas in the rooted layer below, community structure and biomass mainly differed between plant species. This plant effect was also extended into the root-free soil at a depth of 70 cm. In winter, the availability of litter also affected fungal communities in deeper soil layers, suggesting vertical transport processes under fallow conditions. These distinct resource effects indicate diverse ecological niches along the soil profile, comprising specific fungal metacommunities. The recorded responses to both living plants and litter point to a central role of fungi in connecting primary production and decomposition within the plant-soil system.

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.

Ribonucleoprotein (RNP) granules are important posttranscriptional regulators of messenger RNA (mRNA) fate. Several types of RNP granules specifically regulate gene expression during development of multicellular organisms and are commonly referred to as germ granules. The function of germ granules is not entirely understood and probably diverse, but it is generally agreed that one main function is posttranscriptional regulation of gene expression during early development, when transcription is silent. One example is the translational repression of maternally derived mRNAs in oocytes. Here, I hope to show that the need for regulation of gene expression by RNP granules is not restricted to animal development, but plays an equally important role during the development of pathogenic protozoa. Apicomplexa and Trypanosomatidae have complex life cycles with frequent host changes. The need to quickly adapt gene expression to a new environment as well as the ability to suppress translation to survive latencies is critical for successful completion of life cycles. Posttranscriptional gene regulation is not necessarily simpler in protozoa. Apicomplexa surprise with the presence of micro RNA (miRNAs) and upstream open reading frames (µORFs). Trypanosomes have an unusually large repertoire of different RNP granule types. A better understanding of RNP granules in protozoa may help to gain insight into the evolutionary origin of RNP granules: Trypanosomes for example have two types of granules with interesting similarities to animal germ granules. WIREs RNA 2014, 5:263–284. doi: 10.1002/wrna.1207 Conflict of interest: The author has declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

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.

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.

The trypanosome genome is characterized by RNA polymerase II-driven polycistronic transcription of protein-coding genes. Ten to hundreds of genes are co-transcribed from a single promoter; thus, selective regulation of individual genes via initiation is impossible. However, selective responses to external stimuli occur and post-transcriptional mechanisms are thought to account for all temporal gene expression patterns. We show that genes encoding mRNAs that are differentially regulated during the heat-shock response are selectively positioned in polycistronic transcription units; downregulated genes are close to transcription initiation sites and upregulated genes are distant. We demonstrate that the position of a reporter gene within a transcription unit is sufficient to reproduce this effect. Analysis of gene ontology annotations reveals that positional bias is not restricted to stress–response genes and that there is a genome-wide organization based on proximity to transcription initiation sites. Furthermore, we show that the relative abundance of mRNAs at different time points in the cell division cycle is dependent on the location of the corresponding genes to transcription initiation sites. This work provides evidence that the genome in trypanosomes is organized to facilitate co-coordinated temporal control of gene expression in the absence of selective promoters.

To examine the association between characteristics of Stargardt disease and visual acuity (VA), to estimate the longitudinal rate of VA loss, and to identify risk factors for VA loss.Retrospective, multicenter cohort study.A total of 176 patients (332 eyes) with molecularly and clinically confirmed Stargardt disease enrolled from the United States and Europe.Standardized data report forms were used to collect retrospective data on participants' characteristics and best-corrected or presenting VA from medical charts. Linear models with generalized estimating equations were used to estimate the cross-sectional associations, and linear mixed effects models were used to estimate the longitudinal VA loss.Yearly change in VA.The median duration of observation was 3.6 years. At baseline, older age of symptom onset was associated with better VA, and a longer duration of symptoms was associated with worse VA. Longitudinal analysis estimated an average of 0.3 lines loss (P0.0001) per year overall, but the rate varied according to baseline VA: (1) eyes with baseline VA ≥20/25 (N = 53) declined at a rate of approximately 1.0 line per year; (2) eyes with VA between 20/25 and 20/70 (N = 65) declined at a rate of approximately 0.9 lines per year; (3) eyes with VA between 20/70 and 20/200 (N = 163) declined at a rate of 0.2 lines per year; and (4) eyes with VA worse than 20/200 (n = 49) improved at a rate of 0.5 lines per year. Older age of onset was associated with slower VA loss: Patients with onset age30 years showed 0.4 lines slower change of VA per year (P = 0.01) compared with patients with onset age ≤14 years.Given the overall slow rate of VA loss, VA is unlikely to be a sensitive outcome measure for treatment trials of Stargardt disease. However, given the faster decline in younger patients and those with no or mild visual impairment, VA may be a potential outcome measure for trials targeting such subgroups of patients. These observations will need to be assessed in a prospective study bearing in mind the inherent limitations of retrospective datasets.

Soil food webs are important determinants for the carbon flow through terrestrial systems, with the trophic networks between microbes and microfaunal grazers forming the basis for processing plant resources. At an agricultural field site cropped with maize or wheat, plant carbon input to soil was experimentally manipulated by amendment with maize litter. The community structure of dominant micro-food web components, the bacteria, fungi, protozoa and nematodes, was investigated across a depth gradient comprising plough layer, rooted soil below plough horizon, and deeper root free soil. The community composition and diversity within micro-food webs, and the response to resource supply, were assessed in summer, the vegetation period with highest root exudation. In the plough layer amendment with plant residues increased microbial biomass as well as density of fan shaped amoeba morphotypes and of bacterial- and fungal-feeding nematodes. Diversity of food web assemblages was assessed by operational taxonomic units (OTU) for bacteria and fungi, protozoa morphotypes and nematode families. Changes in diversity were either not apparent (fungi, protozoa), negatively related to litter (bacteria) or positively linked to the presence of a specific crop plant (bacteria, nematodes). Based on nematode functional guilds and the related enrichment and structure index, general food web conditions were assigned as nutrient enriched, with a high degree of disturbance, and a dominance of the bacterial energy channel. In sum, litter amendment fostered the abundance but not the diversity of organisms as food webs remained bottom heavy with only small amounts of carbon conserved at higher trophic levels. Food web structure was more affected by the abiotic (soil profile) and biotic (crop plant) environment than by the supply with litter resources.

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 (C org ), extractable organic C (EOC), microbial biomass (C mic ) 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 C org , EOC, C mic and ergosterol pools, respectively. Fungi incorporated maize-derived C to a greater extent than did total soil microbial biomass.

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.

Infection of certain cell types by HIV results in formation of syncytia. This process can be blocked by antibodies or compounds that prevent interaction of viral envelope protein with host cell receptors. Here the authors describe an automated imaging-based assay for inhibitors of cell-cell fusion mediated by interaction of HIV gp120 with CXCR4 coreceptor. The assay quantifies syncytia formation between U87MG astrocytoma cells constitutively expressing CD4/CXCR4 and morphologically distinct Jurkat T lymphoma cells inducibly expressing HIV env. Each cell type was differentially labeled with vital dyes. Fusion was quantified by measuring size, shape, and color of Jurkat cells and Jurkat-harboring cell syncytia. Dose-response experiments with reference inhibitors AMD 3100 and KRH-1636 yielded potencies consistent with those obtained using standard antiviral assays. This assay complements virus-based infectivity assays for identification of inhibitors of membrane fusion events triggered by interaction of HIV gp120 with host CXCR4.

All organisms are able to modulate gene expression in response to internal and external stimuli. Trypanosomes represent a group that diverged early during the radiation of eukaryotes and do not utilise regulated initiation of transcription by RNA polymerase II. Here, the mechanisms present in trypanosomes to alter gene expression in response to stress and change of host environment are discussed and contrasted with those operating in yeast and cultured mammalian cells.

In trypanosomatids, alterations in gene expression in response to intrinsic or extrinsic signals are achieved through post-transcriptional mechanisms. In the last 20 years, research has concentrated on defining the responsible cis-elements in the untranslated regions of several regulated mRNAs. More recently, the focus has shifted towards the identification of RNA-binding proteins that act as trans-acting factors. Trypanosomatids have a large number of predicted RNA-binding proteins of which the vast majority have no orthologues in other eukaryotes. Several RNA-binding proteins have been shown to bind and/or regulate the expression of a group of mRNAs that code for functionally related proteins, indicating the possible presence of co-regulated mRNA cohorts.

Objective: In human astrocytes, restriction of HIV replication involves inhibition of HIV Rev activity. We previously identified a Rev-interacting human protein fragment (16.4.1) that can reduce Rev activity. The 16.4.1 sequence is contained in a group of highly similar host cell proteins, which we call the Risp family. Here we investigate whether the Risp family is connected to HIV replication in astrocytes. Methods: Cell/tissue lysates were analyzed for Risp expression by western blot with various anti-Risp antibodies. The interaction of astrocytic Risp members with Rev was investigated by affinity chromatography. Astrocytes were transfected with expression plasmids containing cDNAs encoding full-length Risp or the isolated 16.4.1 region for Risp overexpression or with siRNAs designed for Risp knock-down. Rev activity was investigated with a Rev-reporter assay. RNA levels were quantified by real-time RT-PCR, HIV Gag levels by p24ELISA. Results: Expression of the Risp family was demonstrated in human brain tissues and astrocytes. Astrocytes were shown to produce Risp family members that interact with Rev. Production of HIV Gag proteins and Rev-dependent RNAs in persistently infected astrocytes increased upon Risp knock-down and decreased upon Risp overexpression. Risp knock-down increased Rev activity and raised proportions of Rev proteins in the nucleus of astrocytes. Conclusion: Our results link the Risp family to restriction of HIV production and inhibition of Rev activity in astrocytes. We conclude that the Risp family represents a novel family of host factors that can control HIV replication and may be important for the containment of HIV infection in brain reservoirs.