Effectiveness and safety of adalimumab in patients with ankylosing spondylitis or psoriatic arthritis and history of anti-tumor necrosis factor therapy

Introduction  

Tumor necrosis factor (TNF) antagonists reduce the signs and symptoms of spondyloarthritides, including ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Our objective was to evaluate the effectiveness and safety of adalimumab, 40 mg every other week, for patients with AS or PsA and prior treatment with infliximab (IFX) and/or etanercept (ETN).     

Annotator: postprocessing software for generating function-based signatures from quantitative mass spectrometry

Mass spectrometry is used to investigate global changes in protein abundance in cell lysates. Increasingly powerful methods of data collection have emerged over the past decade, but this has left researchers with the task of sifting through mountains of data for biologically significant results. Often, the end result is a list of proteins with no obvious quantitative relationships to define the larger context of changes in cell behavior. Researchers are often forced to perform a manual analysis from this list or to fall back on a range of disparate tools, which can hinder the communication of results and their reproducibility. To address these methodological problems, we developed Annotator, an application that filters validated mass spectrometry data and applies a battery of standardized heuristic and statistical tests to determine significance. To address systems-level interpretations, we incorporated UniProt and Gene Ontology keywords as statistical units of analysis, yielding quantitative information about changes in abundance for an entire functional category. This provides a consistent and quantitative method for formulating conclusions about cellular behavior, independent of network models or standard enrichment analyses. Annotator allows for "bottom-up" annotations that are based on experimental data and not inferred by comparison to external or hypothetical models. Annotator was developed as an independent postprocessing platform that runs on all common operating systems, thereby providing a useful tool for establishing the inherently dynamic nature of functional annotations, which depend on results from ongoing proteomic experiments. Annotator is available for download at http://people.cs.uchicago.edu/～tyler/annotator/annotator_desktop_0.1.tar.gz .     

Evidence for introgression in azaleas (Rhododendron; Ericaceae): Chloroplast DNA and morphological variation in a hybrid swarm on Stone Mountain,Georgia

Hybridization and introgression have been suggested by previous workers as important factors in the evolution of the southeastern azaleas. However, because of high levels of intraspecific variation and lack of intensive sampling, documentation of this phenomenon has been difficult. We investigated a population of azaleas on Stone Mountain, Georgia, composed of Rhododendron flammeum, R. canescens, and their interspecific hybrids. After analysis of chloroplast DNA, we found evidence of extensive localized cytoplasmic introgression into R. flammeum. Many individuals in the sample population that are morphologically indistinguishable from R. flammeum possess the chloroplast genome of R. canescens. This suggests that some of the observed variation in some populations of R. flammeum may be due to past introgression from R. canescens.     

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.     

Phosphoprotein profiling by PA-GeLC-MS/MS

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.     

Starch as a sugar reservoir for nematode-induced syncytia

The plant parasitic nematode Heterodera schachtii invades the roots of Arabidopsis thaliana to induce nematode feeding structures in the central cylinder. During nematode development, the parasites feed exclusively from these structures. Thus, high sugar import and specific sugar processing of the affected plant cells is crucial for nematode development. In the present work, we found starch accumulation in nematode feeding structures and therefore studied the expression genes involved in the starch metabolic pathway. The importance of starch synthesis was further shown using the Atss1 mutant line. As it is rather surprising to find starch accumulation in cells characterised by a high nutrient loss, we speculate that starch serves as long- and short-term carbohydrate storage to compensate the staggering feeding behaviour of the parasites.Key words: Heterodera schachtii, Arabidopsis, nematode, starch metabolism, syncytiaThe obligate plant parasitic nematode Heterodera schachtii is entirely dependent on a system of nutrient supply provided by the plant. Host plants—among those the model plant Arabidopsis thaliana—have to endure invasion of second stage juveniles and the establishment of nematode feeding structures in the plant''s vascular cylinder. For induction of the specific feeding structures, the juveniles pierce one single plant cell with their stylet and inject secretions, thus triggering the formation of a syncytium by local cell walls dissolutions.¹ Further, the central vacuole of the syncytial cells disintegrates, nuclei enlarge and many organelles proliferate.¹ About 24 hours after feeding site induction, the nematode juveniles start feeding in repetitive cycles.² Syncytia have previously been described as strong sinks in the plant''s transport system.³ Thus, in the recent years several studies were carried out to discover solute supply to syncytial cells.^4–7 To our present knowledge, syncytia are symplasmically isolated in the first days of nematode development. During that period, the nematodes depend on transport protein activity in the syncytia plasmamembranes. At later stages plasmodesmata appear to open to the phloem elements, facilitating symplasmic transport.Incoming solutes may either be taken up by the feeding nematode or are synthesised and catalysed by the syncytium''s metabolism. Due to the microscopically observable high density of the cytosol¹ and the increased osmotic pressure,⁸ syncytia appear to accumulate high solute concentrations. In fact, significantly increased sucrose levels have been found in syncytia in comparison to non-infected control roots.⁷ In case of high sugar levels, plant cells generally synthesize starch in order to reduce emerging osmotic stress.⁹ The aim of the work of Hofmann et al.,¹⁰ was to elucidate if starch is utilised as carbohydrate storage in nematode-induced syncytia and to study expression of genes involved in starch metabolism with an emphasis on nematode development.Starch levels of nematode induced syncytia and roots of non-infected plants grown on sand/soil culture were measured by high performance liquid chromatography (HPLC). The results showed a high accumulation of starch in syncytia that was steadily decreasing during nematode development. The accumulation of starch could further be localised within syncytial cells by electron microscopy. Based on these results, we studied the gene expression of the starch metabolic pathway by Affymetrix gene chip analysis. About half of the 56 involved genes were significantly upregulated in syncytia compared to the control and only two genes were significantly downregulated. Thus, the high induction of the gene expression is consistent with the high starch accumulation. Finally, we applied an Arabidopsis mutant line lacking starch synthase I expression that has been described previously.¹¹ Starch synthase I was the second highest upregulated gene in syncytia. It catalyses the linkage of ADP-glucose to the non-reducing end of an a-glucan, forming the linear glucose chains of amylopectin. In a nematode infection assay we were able to prove the significant importance of the gene for nematode development.With the presented results, we can unambiguously prove the accumulation of starch and the induction of the gene expression of the starch metabolic pathway in nematode-induced syncytia. The primary question however is: why do syncytia accumulate soluble sugars and starch although their metabolism is highly induced and nematodes withdraw solutes during continuously repeating feeding cycles?One explanation may be found where least expected—in nematode feeding. It is the feeding activity that induced solute import mechanisms into syncytia resulting in a newly formed sink tissue. However, during moulting events to the third, the fourth juvenile stage and to the adult stage nematodes interrupt feeding for about 20 hours.² During this period sugar supply mechanisms will most probably not be altered thus leading to increasing levels of sugars in the syncytium. Starch may serve as short-term carbohydrate buffering sugar excess. Further, starch may serve as long-term carbohydrate storage during nematode development. In the early stages of juvenile development nematodes withdraw considerably small quantities (about 0,8-times the syncytium volume a day).¹² At later stages, nutrient demand increases so that adult fertilised females require 4-times the syncytium volume per day in order to accomplish egg production.¹² Thus, excessive sugar supply in the first days may be accumulated as starch that gets degraded at later stages when more energy is required from the parasites. Consequently, starch reserve serves as both short-term and long-term carbohydrate storage in nematode-induced syncytia in order to buffer changing feeding pattern of the parasites.? Open in a separate windowFigure 1Arabidopsis wild-type Columbia-0 plants were grown in sand/soil culture. Nematode-induced syncytia and non-infected control roots were harvested at 10, 15 and 20 days after inoculation (dai) and starch content was measured as glucose (Glc) equivalents. Values are means ± SE, n = 3. Different letters indicate significant variations (p < 0.05). © ASPBOpen in a separate windowFigure 2Transmission electron microscope picture of a cross-section of a syncytium associated with female fourth stage juvenile (H. schachtii) induced in roots of Arabidopsis. Bar = 2 µm. S, syncytium; Se, sieve tube; arrow, plastid; asterisk, starch granule. © ASPB     

Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study

Introduction  

Improvement of rheumatoid arthritis (RA) during pregnancy has been causatively associated with increased galactosylation of immunoglobulin G (IgG) N-glycans. Since previous studies were small, did not include the postpartum flare and did not study sialylation, these issues were addressed in the present study.     

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.