N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.     

The role of gamma-band activity in the representation of faces: reduced activity in the fusiform face area in congenital prosopagnosia

Background

Congenital prosopagnosia (CP) describes an impairment in face processing that is presumably present from birth. The neuronal correlates of this dysfunction are still under debate. In the current paper, we investigate high-frequent oscillatory activity in response to faces in persons with CP. Such neuronal activity is thought to reflect higher-level representations for faces.

Methodology

Source localization of induced Gamma-Band Responses (iGBR) measured by magnetoencephalography (MEG) was used to establish the origin of oscillatory activity in response to famous and unknown faces which were presented in upright and inverted orientation. Persons suffering from congenital prosopagnosia (CP) were compared to matched controls.

Principal Findings

Corroborating earlier research, both groups revealed amplified iGBR in response to upright compared to inverted faces predominately in a time interval between 170 and 330 ms and in a frequency range from 50–100 Hz. Oscillatory activity upon known faces was smaller in comparison to unknown faces, suggesting a “sharpening” effect reflecting more efficient processing for familiar stimuli. These effects were seen in a wide cortical network encompassing temporal and parietal areas involved in the disambiguation of homogenous stimuli such as faces, and in the retrieval of semantic information. Importantly, participants suffering from CP displayed a strongly reduced iGBR in the left fusiform area compared to control participants.

Conclusions

In sum, these data stress the crucial role of oscillatory activity for face representation and demonstrate the involvement of a distributed occipito-temporo-parietal network in generating iGBR. This study also provides the first evidence that persons suffering from an agnosia actually display reduced gamma band activity. Finally, the results argue strongly against the view that oscillatory activity is a mere epiphenomenon brought fourth by rapid eye-movements (micro saccades).     

Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.     

Proton Pump Inhibitor Intake neither Predisposes to Spontaneous Bacterial Peritonitis or Other Infections nor Increases Mortality in Patients with Cirrhosis and Ascites

Background and Aim

The aim of this study was to assess the impact of proton pump inhibitor (PPI) intake on the development of spontaneous bacterial peritonitis (SBP) or other infections, as well as on mortality, in a thoroughly documented cohort of patients with cirrhosis and ascites.

Patients and Methods

We performed a retrospective analysis of follow-up data from 607 consecutive patients with cirrhosis undergoing their first paracentesis at a tertiary center. A binary logistic regression model investigating the association between PPI intake and SBP at the first paracentesis was calculated. Competing risk analyses and Cox models were used to investigate the effect of PPIs on the cumulative incidence of SBP or other infections and transplant-free survival, respectively. Adjustments were made for age, hepatocellular carcinoma, history of variceal bleeding, varices and model of end-stage liver disease score.

Results

Eighty-six percent of patients were receiving PPIs. After adjusting for potential confounding factors, PPI intake was neither associated with increased SBP prevalence at the first paracentesis (odds ratio (OR):1.11,95% confidence interval (95%CI):0.6–2.06; P = 0.731) nor cumulative incidence of SBP (subdistribution hazard ratio (SHR): 1.38; 95%CI:0.63–3.01; P = 0.42) and SBP or other infections (SHR:1.71; 95%CI:0.85–3.44; P = 0.13) during follow-up. Moreover, PPI intake had no impact on transplant-free survival in both the overall cohort (hazard ratio (HR):0.973,95%CI:0.719–1.317; P = 0.859) as well as in the subgroups of patients without SBP (HR:1.01,95%CI:0.72–1.42; P = 0.971) and without SBP or other infections at the first paracentesis (HR:0.944,95%CI:0.668–1.334; P = 0.742).

Conclusions

The proportion of cirrhotic patients with PPI intake was higher than in previous reports, suggesting that PPI indications were interpreted liberally. In our cohort with a particularly high prevalence of PPI intake, we observed no association between PPIs and SBP or other infections, as well as mortality. Thus, the severity of liver disease and other factors, rather than PPI treatment per se may predispose for infectious complications.     

Here Today,Gone Tomorrow – Adaptation to Change in Memory-Guided Visual Search

Visual search for a target object can be facilitated by the repeated presentation of an invariant configuration of nontargets (‘contextual cueing’). Here, we tested adaptation of learned contextual associations after a sudden, but permanent, relocation of the target. After an initial learning phase targets were relocated within their invariant contexts and repeatedly presented at new locations, before they returned to the initial locations. Contextual cueing for relocated targets was neither observed after numerous presentations nor after insertion of an overnight break. Further experiments investigated whether learning of additional, previously unseen context-target configurations is comparable to adaptation of existing contextual associations to change. In contrast to the lack of adaptation to changed target locations, contextual cueing developed for additional invariant configurations under identical training conditions. Moreover, across all experiments, presenting relocated targets or additional contexts did not interfere with contextual cueing of initially learned invariant configurations. Overall, the adaptation of contextual memory to changed target locations was severely constrained and unsuccessful in comparison to learning of an additional set of contexts, which suggests that contextual cueing facilitates search for only one repeated target location.     

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.     

Experimental demography of the rare Gentianella germanica: seed bank formation and microsite effects on seedling establishment

The persistence of populations of short-lived species requires regular reproduction and seedling establishment. A persistent seed bank can buffer populations against extinction in unfavourable years. We experimentally investigated seed fate in Gentianella germanica, an endangered biennial species characteristic for species-rich nutrient-poor calcareous grasslands in central Europe. We studied the effect of experimental gaps on seedling establishment from sown seeds and the fate of seeds buried in bags over two years. In December 1993 experiments were established at seven calcareous grassland sites in the Swiss Jura mountains. In spring 1994 seedlings emerged in all plots where seeds had been sown, including previously unoccupied patches. This suggests that limited dispersal within sites contributes to small population sizes. Significantly more seedlings emerged at sites with current populations of G. germanica than at unoccupied sites (5.95% vs 3.40%). Because this difference was not explained by germinations from the natural seed bank it indicates differences in habitat quality. Clipping of the vegetation and disturbance of the soil reduced vegetation cover in the following spring and enhanced seedling emergence. In undisturbed plots 4.5% of seeds sown produced a seedling in spring 1994, whereas in plots with clipped vegetation 9.9% and in disturbed plots 12.7% produced seedlings (p>0.01). This suggests that management measures which create gaps in the vegetation (e.g. grazing) could positively influence population size and persistence of G. geymanica. On average, we recovered 7.55% viable seeds after one year of burial in bags, and 4.05% after two years, indicating that G. geymanica has a persistent seed bank. The demographic data indicate that the number of viable seeds in the seed bank exceeds the number of established plants in a population at least by a factor of 20. Restoration of extinct populations of the species from the seed bank may thus be possible if appropriate management measures are taken within a few years.     

Platform-basin transect of a middle to late Jurassic large-scale carbonate platform system (Shotori mountains,Tabas area,east-central Iran)

Summary Following a phase of predominantly siliciclastic sedimentation in the Early and Middle Jurassic, a large-scale, low-latitude carbonate depositional system was established in the northern part of the Tabas Block, part of the central-east Iranian microplate, during the Callovian and persisted until the latest Oxfordian/Early Kimmeridgian. Running parallel to the present eastern block margin, a NNW/SSE-trending carbonate platform developed in an area characterized by reduced subsidence rates (Shotori Swell). The growth of this rimmed, flat-topped barrier platform strongly influenced the Upper Jurassic facies pattern and sedimentary history of the Tabas Block. The platform sediments, represented by the predominantly fine-grained carbonates of the Esfandiar Limestone Formation, pass eastward into slope to basin sediments of the Qal'eh Dokhtar Limestone Formation (platform-derived allochthonites, microbialites, and peri-platform muds). Towards the west, they interfinger with bedded limestones and marlstones (Kamar-e-Mehdi Formation), which were deposited in an extensive shelf lagoon. In a N−S direction, the Esfandiar Platform can be traced for about 170 km, in an E-W direction, the platform extended for at least 35–40 km. The width of the eastern slope of the platform is estimated at 10–15 km, the width of the western shelf lagoon varied considerably (>20–80 km). During the Late Callovian to Middle Oxfordian, the Esfandiar Platform flourished under arid climatic conditions and supplied the slope and basinal areas with large amounts of carbonates (suspended peri-platform muds and gravitational sediments). Export pulses of platform material, e.g. ooids and aggregate grains, into the slope and basinal system are interpreted as highstand shedding related to relative sealevel variations. The high-productivity phase was terminated in the Late Oxfordian when the eastern platform areas drowned and homogeneous deep water marls of the Upper Oxfordian to Kimmeridgian Korond Formation onlapped both the Qal'eh Dokhtar Limestone Formation and the drowned Esfandiar Limestone Formation. Tectonic instability, probably caused by faulting at the margins of the Tabas Block in connection with rotational movements of the east-central Iranian block assemblage, was responsible for the partial drowning of the eastern platform areas. In some areas, relicts of the platform persisted to produce shallow-water sediments into the Kimmeridgian.     

Extending the scope of the controlled logical clock

Event traces are helpful in understanding the performance behavior of parallel applications since they allow the in-depth analysis of communication and synchronization patterns. However, the absence of synchronized clocks on most cluster systems may render the analysis ineffective because inaccurate relative event timings may misrepresent the logical event order and lead to errors when quantifying the impact of certain behaviors or confuse the users of time-line visualization tools by showing messages flowing backward in time. In our earlier work, we have developed a scalable algorithm called the controlled logical clock that eliminates inconsistent inter-process timings postmortem in traces of pure MPI applications, potentially running on large processor configurations. In this paper, we first demonstrate that our algorithm also proves beneficial in computational grids, where a single application is executed using the combined computational power of several geographically dispersed clusters. Second, we present an extended version of the algorithm that—in addition to message-passing event semantics—also preserves and restores shared-memory event semantics, enabling the correction of traces from hybrid applications.