The evolution of the ribosome biogenesis pathway from a yeast perspective

Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.     

Vegetation patterns of floodplain meadows along the climatic gradient at the Middle Elbe River

Central European floodplain meadows are characterised by flooding mainly in winter/spring and dry conditions over the summer. They harbour many rare and endangered plant species. We studied the vegetation of floodplain meadows along the regional climatic gradient of the Middle Elbe River. This gradient exhibits rather continental conditions in the south-east and oceanic conditions in the north-west. We aimed at detecting the influence of climate on the vegetation of floodplain meadows along this gradient.     

Reproductive strategy of the ghost shrimp Callichirus major (Crustacea: Axiidea: Callianassidae) from the southwestern Atlantic: sexual maturity of females,fecundity, egg features,and reproductive output

Reproduction and reproductive investment of females is an intriguing feature in axiidean shrimps. They have a cryptic behavior and great ecological importance in sediment turnover, and recycling of organic material and nutrient. Herein, we describe different aspects of the reproductive biology (size at sexual maturity of females, fecundity, egg characteristics, and reproductive output [RO]) of the ghost shrimp Callichirus major from the southeastern coast of Brazil. Females reached sexual maturity at sizes ≥11.85?mm dorsal oval length. Fecundity increased significantly with female size and varied between 1455 and 9931 eggs (average 4564 eggs). Individual egg volume almost doubled during embryogenesis, and average egg water content increased during the incubation period from 75.0 to 93.3%. The egg mass comprised on average 13.2% of the dry body weight of females. This relatively high RO value is probably compensatory to the relatively low number of ovigerous females in axiidean populations. The few published data on RO values suggest that female investment in reproduction of axiidean shrimps is somewhat higher than in other decapods. The high investment in egg production reflects most likely an effort to maximize the viability of the progeny. Studies with additional congeneric species will clarify whether there is a tendency of axiidean shrimps to have RO values at the upper end of the range reported for decapods.     

Reproductive biology of the sea anemone shrimp Periclimenes rathbunae (Caridea,Palaemonidae, Pontoniinae), from the Caribbean coast of Costa Rica

Caridean shrimps are a highly diverse group and many species form symbiotic relationships with different marine invertebrates. Periclimenes rathbunae is a brightly colored shrimp that lives predominantly in association with sea anemones. Information about the reproductive ecology of the species is scarce. Therefore, we collected 70 ovigerous females inhabiting the sun sea anemone Stichodactyla helianthus in coral reefs from the southern Caribbean coast of Costa Rica. Females produced on average 289 ± 120 embryos. The volume of recently-produced embryos was on average 0.038 mm³, and embryo volume increased by 192% during the incubation period. The average embryo mortality during embryogenesis was 24%. The reproductive output was 0.24 ± 0.094, considerably higher than in many other pontoniine shrimps. Females carrying embryos close to hatching showed fully developed ovaries, suggesting consecutive spawning. We assume that the sheltered habitat, living on sea anemones, allows Periclimenes rathbunae to allocate more energy in embryo production than most other free-living caridean shrimps. This is the first record of Periclimenes rathbunae for Costa Rica.     

Embryo production in the sponge-dwelling snapping shrimp Synalpheus apioceros (Decapoda,Alpheidae) from Bocas del Toro,Panama

Caridean shrimps of the genus Synalpheus are abundant and widely distributed in tropical and subtropical regions, but knowledge of their reproductive biology remains scarce. We report reproductive traits of Synalpheus apioceros from Bocas del Toro, Panama, based on collections in August 2011. The 46 ovigerous females that were analyzed ranged in size from 3.8 to 7.4 mm in carapace length. Fecundity varied between 8 and 310 embryos and increased with female size. Females invested 18.6 ± 10.3% of their body weight in Embryo production. Embryo volume increased considerably (77.2%) during embryogenesis, likely representing water uptake near the end of incubation period. Compared to Synalpheus species with abbreviated or direct development, Synalpheus apioceros produced substantially smaller embryos; however, Synalpheus apioceros seems to have a prolonged larval phase with at least five zoeal stages, which may explain the combination of relatively small and numerous embryos. We did not find nonviable, minute, chalky embryos, previously reported for Synalpheus apioceros specimens obtained from the northwestern Gulf of Mexico, which supports the hypothesis that the production of this type of embryos may be a physiological response of this warm-water species to the temperature decrease near to its latitudinal range limit.     

Preliminary survey of a nemertean crab egg predator,Carcinonemertes, on its host crab,Callinectes arcuatus (Decapoda,Portunidae) from Golfo de Nicoya,Pacific Costa Rica

The possible presence of egg predators in brood masses of portunid crabs from Pacific Central America has not been studied yet. This survey reports the finding of a nemertean crab egg predator on the portunid crab, Callinectes arcuatus, from the Golfo de Nicoya, Pacific Costa Rica. Nemerteans were found in the egg masses of 26 out of the 74 crabs for a prevalence of 35%. The intensity (mean number of worms/ infected crab) was estimated to be 18 with a variance of 1–123 worms/infected crab. No nemerteans were observed either in the 19 Callinectes arcuatus from Golfo Dulce (southern Pacific coast) and the 10 Portunus asper from Herradura-Jaco (central Pacific coast). This nemertean is a member of the genus Carcinonemertes, which has been reported from the Caribbean coast of Panama. However, the encountered Carcinonemertes sp. is the first published finding and report from Costa Rica and Pacific Central America.     

Hypersensitive response to Potato virus Y in potato cultivar Sárpo Mira is conferred by the Ny-Smira gene located on the long arm of chromosome IX

Potato virus Y (PVY, Potyvirus) is the fifth most important plant virus worldwide in terms of economic and scientific impact. It infects members of the family Solanaceae and causes losses in potato, tomato, tobacco, pepper and petunia production. In potato and its wild relatives, two types of resistance genes against PVY have been identified. While Ry genes confer symptomless extreme resistance, Ny genes cause a hypersensitive response visible as local necrosis that may also be able to prevent the virus from spreading under certain environmental conditions. The potato cultivar Sárpo Mira originates from Hungary and is highly resistant to PVY, although the source of this resistance remains unknown. We show that cv. Sárpo Mira reacts with a hypersensitive response leading to necrosis after PVY^NTN infection in detached leaf, whole plant and grafting assays. The hypersensitivity to PVY^NTN segregated amongst 140 individuals of tetraploid progeny of cvs. Sárpo Mira × Maris Piper in a 1:1 ratio, indicating that it was conferred by a single, dominant gene in simplex. Moreover, we identified five DNA markers linked to this trait and located the underlying locus (Ny-Smira) to the long arm of potato chromosome IX. This position corresponds to the location of the Ry _chc and Ny-1 genes for PVY resistance. A simple PCR marker, located 1 cM from the Ny-Smira gene, can be recommended for selection of PVY-resistant progeny of cv. Sárpo Mira.     

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co‐produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH‐9048 and TcsL‐9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN‐TcsH‐9048 and rN‐TcdA‐10463 glucosylated preferably Rho‐GTPases but also Ras‐GTPases to some extent. In this respect, rN‐TcsH‐9048 and rN‐TcdA‐10463 differ from the respective full‐length TcsH‐9048 and TcdA‐10463, which exclusively glucosylate Rho‐GTPases. rN‐TcsL‐9048 and full length TcsL‐9048 glucosylate both Rho‐ and Ras‐GTPases, whereas rN‐TcdB‐10463 and full length TcdB‐10463 exclusively glucosylate Rho‐GTPases. Vero cells treated with full length TcsH‐9048 or TcdA‐10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho‐GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto‐proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB‐1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro‐apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118‐paxillin and of pS144/141‐PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.     

Use of 18F-Fluorodeoxyglucose Positron Emission Tomography–Computed Tomography to Aid in Diagnosing Intestinal Adenocarcinoma in 2 Rhesus Macaques (Macaca mulatta)

Two aged female rhesus macaques (Macaca mulatta) presented with weight loss and intermittent inappetence. The signalment and constellation of clinical signs led clinicians to suspect the presence of intestinal adenocarcinoma. Because of each animal''s advanced age and inconclusive radiographic findings, a noninvasive diagnostic tool was preferred over exploratory laparotomy to assist in determining a diagnosis. Consequently, 2-[¹⁸F]fluoro-2-deoxy-d-glucose (FDG) positron emission tomography–CT (FDG-PET–CT) was chosen to aid in confirming a suspicion of gastrointestinal adenocarcinoma in both animals. FDG is a glucose analogue labeled with fluorine-18 and is taken up by highly metabolically active cells, as observed in many cancers. Tomography revealed an annular constriction of the small intestine with focal FDG uptake in one animal, and an FDG avid transmural mass in the ascending colon of the second animal. Necropsy later confirmed both sites to be adenocarcinomas. This report supports the use of FDG-PET–CT as an adjunct to conventional radiography in the diagnosis of intestinal adenocarcinoma in nonhuman primates.Abbreviations: FDG, 2-[¹⁸F]fluoro-2-deoxy-d-glucose; PET, positron emission tomographyThe noninvasive imaging modality 2-[¹⁸F]fluoro-2-deoxy-d-glucose positron emission tomography-computed tomography (FDG-PET–CT) is used primarily for the detection and staging of cancer and for the assessment of therapeutic response.²⁸ This modality also is useful for the identification of some infectious and inflammatory diseases, as demonstrated in the recent report of intense FDG uptake in a case of pneumonia in a miniature pig.¹⁴ Furthermore, FDG-PET–CT has been helpful in the detection and monitoring of inflammatory bowel disease and complicating infections of the gastrointestinal tract.²⁸PET detects the three-dimensional distribution of radioactivity based on the annihilation photons emitted by radiotracers labeled with positron-emitting radionuclides, such as ¹⁸F-labeled FDG. In this manner, PET enables noninvasive assessment of biochemical processes. In contrast, CT uses X-rays to generate high-resolution images, allowing clear visualization of anatomic structures. In PET–CT, both the multidetector CT apparatus and the PET detectors are mounted in the same scanner, one behind the other. The PET data are superimposed on the CT data (coregistration), enabling precise anatomic localization of FDG activity.²⁹ Combining PET imaging with CT allows the fusion of functional and anatomic information acquired almost simultaneously, facilitating the visualization and localization of metabolic information noninvasively.Intestinal adenocarcinoma is a substantial cause of morbidity and mortality in aged rhesus macaques.^25,26,30 Rhesus macaques often are maintained on long term-studies, resulting in a population of animals exhibiting morbidities associated with advanced age.^20,25,26 As the age of the animals in our care advances, so too must our diagnostic capabilities advance. Here we report the use of a noninvasive method, FDG-PET–CT, to support the diagnosis of intestinal adenocarcinoma in 2 aged rhesus macaques. We describe the physics of PET, biochemical rationale for the use of FDG, and pitfalls of interpretation.     

The Interplay of Light and Oxygen in the Reactive Oxygen Stress Response of Chlamydomonas reinhardtii Dissected by Quantitative Mass Spectrometry

Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox^II was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.Life originated in an environment in which the atmosphere was reducing. More than 2.2 Gyr ago, photosynthetic bacteria managed to extract electrons from water, thereby releasing oxygen (O₂) as a side product (1). Although molecular O₂ is a triplet state (³O₂), and is thus kinetically inhibited, its related reactive oxygen species (ROS)¹, i.e., superoxide (O₂^•−), peroxides (ROOR), singlet oxygen (¹O₂), and hydroxyl radicals (HO^•) are not. Nevertheless, molecular O₂ itself oxidizes biomolecules, for example, thiol groups, albeit at a much slower rate. The fundamental change in environment and the appearance of O₂ and ROS triggered the biggest mass extinction ever seen on Earth (2, 3). Soon after, the much more efficient O₂ based metabolism (compared with fermentation) lead to an evolutionary explosion (4). Today, cells obtain energy from reduced organic molecules through O₂ based respiration.In the past ROS were associated with cellular stress but strong evidence points toward a cellular ROS network that keeps ROS production and ROS scavenging in tight balance to ensure the maintenance of the cellular redox homeostasis and protection against ROS stress (5, 6). An imbalance in this network has been associated with a wide array of human diseases such as cancer (7), neurodegeneration (8), Keshan disease (9), and many others (see also review (6)), although arguments have been brought forward that the origin of some diseases is not directly linked to ROS and that ROS are more likely to be the result of deteriorating cells (10). In any case, the cellular ROS network response to ROS stress is implicated in the progress of these diseases and understanding the network dynamics will have a significant impact in medicine.Equally important, reduced ROS capacity or imbalance in the ROS network results in decreased crop yields and simple attempts to increase production yields by increasing ROS scavenging capacities in plants failed because those plants lost their ability to mount a defense against pathogens efficiently by the hypersensitive reaction (11), which implicates intended localized high yield ROS production. On the other hand Chang et al. could show that the knock-out of glutathione peroxidase 7 (gpx7), i.e., reducing ROS scavenging capacity, leads to an increased pathogen resistance but, unfortunately, to an increased photosensitivity as well (12), thus resulting in reduced crop production. The quintessence is that plants require the ability to produce sufficient amounts of ROS as part of their defense mechanism yet require some ROS scavenging capacity because photosynthetic growth inevitably produces damaging ROS. In order to effectively mount a hypersensitivity defense reaction, the ROS scavenging capacities have to be suppressed. Thus understanding the ROS network is an important global issue in the light of hunger in some parts of the world and the need for biofuels. Elucidating the key players of the ROS network will allow high production crop plants to be designed.It seems clear that the ROS network, its dynamics and homeostasis are poorly understood. Understanding how to evaluate the ROS balance and how to restore ROS balance within a cell would have a strong impact on a medical and agricultural level. To put it in the words of Barry Halliwell: “the likely clinical value of ‘antioxidant therapy’ will depend on how well the exact role of reactive oxygen species,” i.e., the ROS network, “is known” (13).ROS can be divided into two classes, i.e., H₂O₂ and ¹O₂ based ones. Especially in plants, algae, and cyanobacteria, it is now widely accepted that the signaling pathways of H₂O₂ (14) and ¹O₂ (15) are complex and entangled (16, 17) simply through the nature of their production, i.e., via an active photosynthetic electron transport chain. However, there have been reports that clearly show the independence of H₂O₂ and ¹O₂ mediated responses (see e.g. (18, 19)). In Arabidopsis thaliana the ROS network, in particular the ¹O₂ aspect has been widely studied, but comprehensive proteomic studies are still required. The A. thaliana flu mutant was used to reveal ¹O₂ related retrograde signaling. The flu mutant accumulates protochlorophyllide when grown in the dark, and seedlings bleach and die whereas mature plants stop growing when transferred into light (20). ¹O₂ production yielded an induction of distinct genes and these differed significantly from genes induced by H₂O₂ (15). Apel and co-workers identified the chloroplast localized EXECUTER1/2 proteins as key players in ¹O₂ retrograde signaling (18, 21), highlighting that specific ¹O₂ induced signals trigger programmed cell death (PCD) rather than ROS induced damage. A flu-like gene (flp) was identified in Chlamydomonas reinhardtii, and its gene product FLP in its two splicing variants was shown to be involved in the chlorophyll biosynthesis (22). Regulation of FLPs were suggested to occur via light and retrograde plastid signals (22). The specific ¹O₂ signaling mechanism in A. thaliana was further extended by Ramel et al. (23). The authors could show that ¹O₂ induced damage to β-carotene, a major component in a ROS defense strategy, yields β-cyclocitral, which when produced and applied exogenously triggers a selective ¹O₂ response, similar to the one reported by Apel and co-workers when describing the effects of the flu mutant (15, 18, 21). However, the signaling pathways involving EXECUTER and β-cyclocitral show more and more independent features (see e.g. Lundquist et al. (24)).ROS production is an inevitable part of the oxygenic photosynthesis and thus can be controlled noninvasively by light intensities. This is why plants, algae, and cyanobacteria offer a unique opportunity to investigate the ROS network. However, in plants the majority of ROS is produced in the chloroplast requiring O₂ as educt and the presence of light. Therefore, careful separation of the light, O₂, and ROS responses is required. As a consequence, simple high light/low light comparisons are overshadowed by additional ROS production, and vice versa. A classical example is HSP70A in C. reinhardtii, which was originally reported to be light regulated (25) and later proven to be regulated by ROS (19), via two promoters that react specifically on H₂O₂ and ¹O₂, to be precise.We have devised an experimental setup, which allows the ROS, high light/low light (HL/LL) and aerobic/anaerobic (AR/AN) responses to be dissected on a proteome level using metabolic labeling and quantitative proteomics. We used an interlinked experimental setup that connects all four possible treatments in such a way that each treatment is compared with two other treatments. This offers a strong internal control because the changes in protein levels comparing two not directly connected treatments can be measured by two independent estimates. MS data was analyzed employing high quality retention time alignment to increase overall confidence in protein quantification, increase protein sequence coverage and increase coverage of all conditions. PyGCluster, a novel hierarchical clustering approach (26) was used to identify communities of proteins that are coregulated. Five communities/expression profiles are discussed: a) light and O₂ dependent induction, i.e., potential ROS related regulations, b) a novel regulation type, which shows induction of protein expression influenced additively by light and O₂, but with light as the obligatory first step, c) light related induction (O₂ independent), d) light dependent repression (O₂ independent), and e) light dependent repression in the absence of O₂, which might be a regulation linked to feedback inhibition by for example, molecules that are normally damaged by ROS.