Transcription factor WOX11 modulates tolerance to cyst nematodes via adventitious lateral root formation.

The transcription factor WUSCHEL-RELATED HOMEOBOX 11 (WOX11) in Arabidopsis (Arabidopsis thaliana) initiates the formation of adventitious lateral roots upon mechanical injury in primary roots. Root-invading nematodes also induce de novo root organogenesis leading to excessive root branching, but it is not known if this symptom of disease involves mediation by WOX11 and if it benefits the plant. Here, we show with targeted transcriptional repression and reporter gene analyses in Arabidopsis that the beet cyst nematode Heterodera schachtii activates WOX11-mediated adventitious lateral rooting from primary roots close to infection sites. The activation of WOX11 in nematode-infected roots occurs downstream of jasmonic acid-dependent damage signaling via ETHYLENE RESPONSE FACTOR109, linking adventitious lateral root formation to nematode damage to host tissues. By measuring different root system components, we found that WOX11-mediated formation of adventitious lateral roots compensates for nematode-induced inhibition of primary root growth. Our observations further demonstrate that WOX11-mediated rooting reduces the impact of nematode infections on aboveground plant development and growth. Altogether, we conclude that the transcriptional regulation by WOX11 modulates root system plasticity under biotic stress, which is one of the key mechanisms underlying the tolerance of Arabidopsis to cyst nematode infections.

NLRscape: an atlas of plant NLR proteins.

NLRscape is a webserver that curates a collection of over 80 000 plant protein sequences identified in UniProtKB to contain NOD-like receptor signatures, and hosts in addition a number of tools aimed at the exploration of the complex sequence landscape of this class of plant proteins. Each entry gathers sequence information, domain and motif annotations from multiple third-party sources but also in-house advanced annotations aimed at addressing caveats of the existing broad-based annotations. NLRscape provides a top-down perspective of the NLR sequence landscape but also services for assisting a bottom-up approach starting from a given input sequence. Sequences are clustered by their domain organization layout, global homology and taxonomic spread-in order to allow analysis of how particular traits of an NLR family are scattered within the plant kingdom. Tools are provided for users to locate their own protein of interest in the overall NLR landscape, generate custom clusters centered around it and perform a large number of sequence and structural analyses using included interactive online instruments. Amongst these, we mention: taxonomy distribution plots, homology cluster graphs, identity matrices and interactive MSA synchronizing secondary structure and motif predictions. NLRscape can be found at: https://nlrscape.biochim.ro/.

The Arabidopsis transcription factor TCP9 modulates root architectural plasticity, reactive oxygen species-mediated processes, and tolerance to cyst nematode infections.

Infections by root-feeding nematodes have profound effects on root system architecture and consequently shoot growth of host plants. Plants harbor intraspecific variation in their growth responses to belowground biotic stresses by nematodes, but the underlying mechanisms are not well understood. Here, we show that the transcription factor TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR-9 (TCP9) modulates root system architectural plasticity in Arabidopsis thaliana in response to infections by the endoparasitic cyst nematode Heterodera schachtii. Young seedlings of tcp9 knock-out mutants display a significantly weaker primary root growth inhibition response to cyst nematodes than wild-type Arabidopsis. In older plants, tcp9 reduces the impact of nematode infections on the emergence and growth of secondary roots. Importantly, the altered growth responses by tcp9 are most likely not caused by less biotic stress on the root system, because TCP9 does not affect the number of infections, nematode development, and size of the nematode-induced feeding structures. RNA-sequencing of nematode-infected roots of the tcp9 mutants revealed differential regulation of enzymes involved in reactive oxygen species (ROS) homeostasis and responses to oxidative stress. We also found that root and shoot growth of tcp9 mutants is less sensitive to exogenous hydrogen peroxide and that ROS accumulation in nematode infection sites in these mutants is reduced. Altogether, these observations demonstrate that TCP9 modulates the root system architectural plasticity to nematode infections via ROS-mediated processes. Our study further points at a novel regulatory mechanism contributing to the tolerance of plants to root-feeding nematodes by mitigating the impact of belowground biotic stresses.

Preclinical models versus clinical renal ischemia reperfusion injury: A systematic review based on metabolic signatures.

Despite decennia of research and numerous successful interventions in the preclinical setting, renal ischemia reperfusion (IR) injury remains a major problem in clinical practice, pointing toward a translational gap. Recently, two clinical studies on renal IR injury (manifested either as acute kidney injury or as delayed graft function) identified metabolic derailment as a key driver of renal IR injury. It was reasoned that these unambiguous metabolic findings enable direct alignment of clinical with preclinical data, thereby providing the opportunity to elaborate potential translational hurdles between preclinical research and the clinical context. A systematic review of studies that reported metabolic data in the context of renal IR was performed according to the PRISMA guidelines. The search (December 2020) identified 35 heterogeneous preclinical studies. The applied methodologies were compared, and metabolic outcomes were semi-quantified and aligned with the clinical data. This review identifies profound methodological challenges, such as the definition of IR injury, the follow-up time, and sampling techniques, as well as shortcomings in the reported metabolic information. In light of these findings, recommendations are provided in order to improve the translatability of preclinical models of renal IR injury.

Biomarkers to identify Mycobacterium tuberculosis infection among borderline QuantiFERON results.

BACKGROUND: Screening for tuberculosis (TB) infection often includes QuantiFERON-TB Gold Plus (QFT) testing. Previous studies showed that two-thirds of patients with negative QFT results just below the cut-off, so-called borderline test results, nevertheless had other evidence of TB infection. This study aimed to identify a biomarker profile by which borderline QFT results due to TB infection can be distinguished from random test variation. METHODS: QFT supernatants of patients with a borderline (≥0.15 and <0.35 IU·mL-1), low-negative (<0.15 IU·mL-1) or positive (≥0.35 IU·mL-1) QFT result were collected in three hospitals. Bead-based multiplex assays were used to analyse 48 different cytokines, chemokines and growth factors. A prediction model was derived using LASSO regression and applied further to discriminate QFT-positive Mycobacterium tuberculosis-infected patients from borderline QFT patients and QFT-negative patients RESULTS: QFT samples of 195 patients were collected and analysed. Global testing revealed that the levels of 10 kDa interferon (IFN)-γ-induced protein (IP-10/CXCL10), monokine induced by IFN-γ (MIG/CXCL9) and interleukin-1 receptor antagonist in the antigen-stimulated tubes were each significantly higher in patients with a positive QFT result compared with low-negative QFT individuals (p<0.001). A prediction model based on IP-10 and MIG proved highly accurate in discriminating patients with a positive QFT (TB infection) from uninfected individuals with a low-negative QFT (sensitivity 1.00 (95% CI 0.79-1.00) and specificity 0.95 (95% CI 0.74-1.00)). This same model predicted TB infection in 68% of 87 patients with a borderline QFT result. CONCLUSIONS: This study was able to classify borderline QFT results as likely infection-related or random. These findings support additional laboratory testing for either IP-10 or MIG following a borderline QFT result for individuals at increased risk of reactivation TB.

Metabolic needs of the kidney graft undergoing normothermic machine perfusion.

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed.

PR3-ANCAs predict relapses in ANCA-associated vasculitis patients after rituximab.

BACKGROUND: The primary challenge of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patient care is the early detection of relapses to prevent organ damage and increase survival. Potential biomarkers for relapses are ANCA and B cells, but their predictive value is a matter of debate. Therefore this study investigated how ANCA and B-cell status related to relapses in AAV patients treated with rituximab (RTX) as remission induction (RI). METHODS: This single-centre cohort study identified 110 ANCA-positive AAV patients treated with RTX between 2006 and 2018. Serial ANCA, CD19+ B-cell status and relapses were assessed >2 years. RESULTS: Patients (31/110) relapsed within 2 years after RTX RI treatment. Patients who achieved and maintained PR3-ANCA negativity (n = 29) had few relapses (3%), while persistent proteinase 3 (PR3)-ANCA positivity (n = 49) and reappearance of PR3-ANCAs (n = 10) associated significantly with more relapses (37%, P = 0.002 and 50%, P = 0.002). Patients with incomplete B-cell depletion (n = 11) had significantly more relapses (54%) as compared with patients with B-cell depletion [n = 76 (26%), P = 0.02]. Also, patients with repopulation of B cells (n = 58) had significantly more relapses (41%) as compared with patients without B-cell repopulation [n = 27 (15%), P = 0.03]. Overall, the absence of PR3- or myeloperoxidase (MPO)-ANCA positivity was highly predictive for remaining relapse-free. In PR3-ANCA-positive patients, 96% of the relapses occurred with persistent or reappearance of PR3-ANCAs and 81% with B-cell repopulation. In MPO-ANCA-positive patients, all relapses were restricted to patients with persistent MPO-ANCAs and B-cell repopulation. CONCLUSIONS: Upon RI treatment with RTX in AAV patients, ANCA and B-cell status were predictive of the majority of relapses and specifically their absence strongly predicted a relapse-free status. Therefore the implementation of ANCA and B-cell monitoring could guide therapeutic decision-making to prevent relapses in AAV patients treated with RTX.

Long-term effects of combined B-cell immunomodulation with rituximab and belimumab in severe, refractory systemic lupus erythematosus: 2-year results.

BACKGROUND: Anti-CD20 B-cell depletion has not shown superior efficacy to standard immunosuppression in patients with systemic lupus erythematosus (SLE). Besides trial design, potential explanations are incomplete B-cell depletion in relation to substantial surges in B-cell-activating factor (BAFF). To improve B-cell targeting strategies, we conducted the first study in SLE patients aimed at investigating immunological effects and feasibility of combining rituximab (RTX; anti-CD20) and belimumab (BLM; anti-BAFF). METHODS: Reported is the long-term follow-up of a Phase 2 proof-of-concept study in 15 patients with SLE including 12 (80%) with lupus nephritis (LN). RESULTS: In 10/15 (67%) patients, a clinical response was observed by achievement of lupus low disease activity state, of which 8 (53%) continued treatment (BLM + ≤7.5 mg prednisolone) for the complete 2 years of follow-up. Five patients (33%) were referred to as 'non-responders' due to persistent LN, major flare or repetitive minor flares. Out of 12 LN patients, 9 (75%) showed a renal response including 8 (67%) complete renal responders. All anti-dsDNA+ patients converted to negative, and both anti-C1q and extractable nuclear antigen autoantibodies showed significant reductions. CD19+ B cells showed a median decrease from baseline of 97% at 24 weeks, with a persistent reduction of 84% up to 104 weeks. When comparing responders with non-responders, CD20+ B cells were depleted significantly less in non-responders and double-negative (DN) B cells repopulated significantly earlier. CONCLUSIONS: Combined B-cell targeted therapy with RTX and BLM prevented full B-cell repopulation including DN B cells, with concomitant specific reduction of SLE-relevant autoantibodies. The observed immunological and clinical benefits in a therapy-refractory SLE population prompt further studies on RTX + BLM.

Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia reperfusion injury.

Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways.

The TIR-NB-LRR pair DSC1 and WRKY19 contributes to basal immunity of Arabidopsis to the root-knot nematode Meloidogyne incognita.

BACKGROUND: Root-knot nematodes transform vascular host cells into permanent feeding structures to withdraw nutrients from the host plant. Ecotypes of Arabidopsis thaliana can display large quantitative variation in susceptibility to the root-knot nematode Meloidogyne incognita, which is thought to be independent of dominant major resistance genes. However, in an earlier genome-wide association study of the interaction between Arabidopsis and M. incognita we identified a quantitative trait locus harboring homologs of dominant resistance genes but with minor effect on susceptibility to the M. incognita population tested. RESULTS: Here, we report on the characterization of two of these genes encoding the TIR-NB-LRR immune receptor DSC1 (DOMINANT SUPPRESSOR OF Camta 3 NUMBER 1) and the TIR-NB-LRR-WRKY-MAPx protein WRKY19 in nematode-infected Arabidopsis roots. Nematode infection studies and whole transcriptome analyses using the Arabidopsis mutants showed that DSC1 and WRKY19 co-regulate susceptibility of Arabidopsis to M. incognita. CONCLUSION: Given the head-to-head orientation of DSC1 and WRKY19 in the Arabidopsis genome our data suggests that both genes may function as a TIR-NB-LRR immune receptor pair. Unlike other TIR-NB-LRR pairs involved in dominant disease resistance in plants, DSC1 and WRKY19 most likely regulate basal levels of immunity to root-knot nematodes.

A reverse translational study on the effect of rituximab, rituximab plus belimumab, or bortezomib on the humoral autoimmune response in SLE.

OBJECTIVES: SLE is a severe autoimmune disease characterized by autoreactive B cells and IC formation, which causes systemic inflammation. B cell-targeted therapy could be a promising treatment strategy in SLE patients; nevertheless, randomized clinical trials have not always been successful. However, some groups have demonstrated beneficial effects in severe SLE patients with off-label rituximab (RTX) with belimumab (BLM), or bortezomib (BTZ), which targeted different B cells subsets. This study assembled sera from SLE cohorts treated with RTX+BLM (n = 15), BTZ (n = 11) and RTX (n = 16) to get an in-depth insight into the immunological effects of these therapies on autoantibodies and IC formation. METHODS: Autoantibodies relevant for IC formation and the avidity of anti-dsDNA were determined by ELISA. IC-mediated inflammation was studied by complement levels and ex vivo serum-induced neutrophil extracellular trap formation. RESULTS: Reductions in autoantibodies were observed after all approaches, but the spectrum differed depending upon the treatment. Specifically, only RTX+BLM significantly decreased anti-C1q. Achieving seronegativity of ≥1 autoantibody, specifically anti-C1q, was associated with lower disease activity. In all SLE patients, the majority of anti-dsDNA autoantibodies had low avidity. RTX+BLM significantly reduced low-, medium- and high-avidity anti-dsDNA, while RTX and BTZ only significantly reduced medium avidity. IC-mediated inflammation, measured by C3 levels and neutrophil extracellular trap formation, improved after RTX+BLM and RTX but less after BTZ. CONCLUSION: This study demonstrated the impact of different B cell-targeted strategies on autoantibodies and IC formation and their potential clinical relevance in SLE.

Trends in diagnostic methods and treatment of latent tuberculosis infection in a tertiary care center from 2000 to 2017.

Screening for latent tuberculosis infection (LTBI) is indicated before immunosuppressive therapies but is complicated by lack of a gold standard and limited by, e.g., immunosuppression. This study aimed to investigate a series of patients diagnosed with LTBI during screening before immunosuppressive therapy, describing how the use of diagnostic tests and treatment evolved over time. This retrospective cohort study included all individuals diagnosed with LTBI during screening before intended immunosuppressive therapy in a tertiary care hospital between January 2000 and December 2017. Evidence for LTBI, including history, tuberculin skin test (TST), QuantiFERON (QFT) result and suggestive lesions on chest radiography (CXR), and CT scan if available, was analyzed. The study included 295 individuals with LTBI, with median follow-up of 3.8 years (IQR 1.7-7.4 years). During screening, TST, QFT, and CXR were positive in 80.8%, 53.4%, and 22.7%, respectively. Chest CT revealed lesions associated with past tuberculosis infection in around 70%, significantly more frequent than CXR. In patients diagnosed with LTBI, we observed that the use of TST declined over time whereas the use of QFT increased, and that isoniazid was replaced with rifampicin as preferential treatment. Preventive treatment was started in 82.3%, of whom 88.6% completed treatment. During follow-up, no individuals developed active tuberculosis. The diagnosis of LTBI was based on history, TST, QFT, and/or CXR in nearly every possible combination, but mostly on TST and QFT. The most striking trends were the decreased use of TST, increased use of QFT, and the replacement of isoniazid with rifampicin for treatment.

Metabolic events in HIV-infected patients using abacavir are associated with erythrocyte inosine triphosphatase activity.

Objectives: Abacavir use has been associated with an increased risk of cardiovascular disease (CVD) and metabolic events in HIV-infected patients, although this finding was not consistently found. It is unclear whether abacavir only increases this risk in subpopulations of HIV-infected patients. It may be hypothesized that inosine 5'-triphosphate pyrophosphohydrolase (ITPase), an enzyme involved in the metabolism of purine analogues used in HIV treatment, plays a role in the risk of CVD and metabolic events in HIV-infected patients. Methods: ITPase activity and ITPA genotype were determined in 393 HIV-infected patients. ITPase activity <4 mmol IMP/mmol Hb/h was considered decreased. ITPA polymorphisms tested were: c.94C>A (rs1127354) and c.124 + 21A>C (rs7270101). ORs were determined using generalized estimating equation models for developing CVD in patients who had ever been exposed to abacavir, tenofovir or didanosine and for developing metabolic events in patients currently using these drugs. Results: In patients using abacavir, metabolic events were associated with ITPase activity. No association was demonstrated for tenofovir or didanosine. The OR for metabolic events was 3.11 in patients using abacavir with normal ITPase activity (95% CI 1.34-7.21; P = 0.008) compared with patients with decreased ITPase activity [adjusted for age, BMI, cumulative duration of combination ART (cART) use and the use of PI and NNRTI]. CVD was not associated with ITPase activity or ITPA genotype. Conclusions: This study shows, for the first time, that ITPase activity is associated with the occurrence of metabolic events in patients using abacavir. Further studies are needed to confirm this association and to elucidate the possible mechanism.

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.

Neutrophil extracellular traps (NETs) are auto-antigenic strands of extracellular DNA covered with myeloperoxidase (MPO) and proteinase3 (PR3) that can be a source for the formation of anti-neutrophil cytoplasmic autoantibodies (ANCAs). The presence of NETs was recently demonstrated in renal tissue of patients with ANCA-associated vasculitis (AAV). NET formation was enhanced in AAV, suggesting that MPO-ANCA could trigger NET formation, supporting a vicious circle placing NETs in the center of AAV pathogenesis. Here we investigated NET formation in 99 patients with AAV by a novel highly sensitive and automated assay. There was a significant excess of ex vivo NET formation in both MPO-ANCA- and PR3-ANCA-positive patients with AAV compared to healthy individuals. Excessive NET formation did not correlate with serum ANCA levels. Likewise, immunoglobulin G depletion had no effect on excessive NET formation in patients with AAV, indicating an ANCA-independent process. Next, we explored the relation of excessive NET formation to clinical disease in ten patients with AAV and showed that excessive NET formation was predominantly found during active disease, more so than during remission. Excessive NET formation was found in patients with AAV hospitalized for disease relapse but not during severe infection. Thus, excessive NET formation in AAV is independent of ANCA, and an excess of ex vivo NET formation was related to active clinical disease in patients with AAV and a marker of autoimmunity rather than infection.

The NET-effect of combining rituximab with belimumab in severe systemic lupus erythematosus.

OBJECTIVE: In systemic lupus erythematosus (SLE) patients, excessive formation of neutrophil extracellular traps (NETs) is observed and their degradation is impaired. In vitro, immune complexes (ICx) trigger NET formation while NET-derived DNA is a postulated autoantigen for anti-nuclear autoantibodies (ANAs), found in SLE. Based on these self-perpetuating mechanisms in SLE, this study investigates whether interfering with ICx formation using a combination of rituximab (RTX) and belimumab (BLM) could decrease NET formation and ameliorate disease. METHODS: A phase 2A, open-label, single arm proof-of-concept study was performed wherein 16 SLE patients with severe, refractory disease were treated with a combination of CD20-mediated B-cell depletion with rituximab and sustained inhibition of B-cell activating factor BlyS with belimumab. Besides safety, the study's endpoints were chosen to address the concept of autoantibodies in relation to excessive NET formation. RESULTS: We demonstrated a surge of BlyS levels upon RTX-mediated B-cell depletion which was abrogated by subsequent BLM treatment. As such, therapeutic intervention with RTX + BLM led to specific reductions in ANAs and regression of excessive NET formation. RTX + BLM appeared to be safe and achieved clinically significant responses: low lupus disease activity state was achieved in 10 patients, renal responses in 11 patients and concomitant immunosuppressive medication was tapered in 14 out of the 16 patients. CONCLUSIONS: This study provides novel insights into clinical beneficence of reducing excessive NET formation in SLE by therapeutic targeting ANA production with RTX + BLM. Altogether putting forward a new treatment concept that specifically ameliorates underlying SLE pathophysiology. TRIAL REGISTRATION: ClinicalTrials.gov NCT02284984.

Genome-wide association mapping of the architecture of susceptibility to the root-knot nematode Meloidogyne incognita in Arabidopsis thaliana.

Susceptibility to the root-knot nematode Meloidogyne incognita in plants is thought to be a complex trait based on multiple genes involved in cell differentiation, growth and defence. Previous genetic analyses of susceptibility to M. incognita have mainly focused on segregating dominant resistance genes in crops. It is not known if plants harbour significant genetic variation in susceptibility to M. incognita independent of dominant resistance. To study the genetic architecture of susceptibility to M. incognita, we analysed nematode reproduction on a highly diverse set of 340 natural inbred lines of Arabidopsis thaliana with genome-wide association mapping. We observed a surprisingly large variation in nematode reproduction among these lines. Genome-wide association mapping revealed four quantitative trait loci (QTLs) located on chromosomes 1 and 5 of A. thaliana significantly associated with reproductive success of M. incognita, none of which harbours typical resistance gene homologues. Mutant analysis of three genes located in two QTLs showed that the transcription factor BRASSINAZOLE RESISTANT1 and an F-box family protein may function as (co-)regulators of susceptibility to M. incognita in Arabidopsis. Our data suggest that breeding for loss-of-susceptibility, based on allelic variants critically involved in nematode feeding, could be used to make crops more resilient to root-knot nematodes.

Sequence Exchange between Homologous NB-LRR Genes Converts Virus Resistance into Nematode Resistance, and Vice Versa.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

To what extent do autoantibodies help to identify high-risk patients in systemic sclerosis?

OBJECTIVES: To evaluate the additive value of autoantibodies in identifying systemic sclerosis (SSc) patients with high complication risk. METHODS: Patients entering the Combined Care In SSc cohort, Leiden University Medical Centre between April 2009 and May 2016 were included. Subgroups of patients were determined using hierarchical clustering, performed on Principal Component Analysis scores, 1) using baseline data of demographic and clinical variables only and 2) with additional use of antibody status. Disease-risk within subgroups was assessed by evaluating 5-year mortality rates. Clinical and autoantibody characteristics of obtained subgroups were compared. RESULTS: In total 407 SSc patients were included, of which 91% (n=371) fulfilled ACR/EULAR 2013 criteria for SSc. Prevalences of autoantibodies were: anti-centromere 37%, anti-topoisomerase (ATA) 24%, anti-RNA polymerase III 5%, anti-fibrillarin 4% and anti-Pm/Scl 5%. Clinical cluster analysis identified 4 subgroups, with two subgroups showing higher than average mortality (resp. 17% and 7% vs. total group mortality of 4%). ATA-positivity ranged from 10 to 21% in low-risk groups and from 30 to 49% among high-risk groups. Adding autoantibody status to the cluster process resulted in 5 subgroups with 3 showing higher than average mortality. Still, 22% of ATA- positive patients were clustered into a low-risk subgroup, while the total number of patients stratified to a high-risk subgroup increased. CONCLUSIONS: Autoantibodies only partially contribute to risk-stratification and clinical subsetting in SSc. The current findings confirm that not all ATA-positive patients have worse prognosis and as such, additional biomarkers are needed to guide clinical follow-up in SSc.

The hypoxanthine-xanthine oxidase axis is not involved in the initial phase of clinical transplantation-related ischemia-reperfusion injury.

The hypoxanthine-xanthine oxidase (XO) axis is considered to be a key driver of transplantation-related ischemia-reperfusion (I/R) injury. Whereas interference with this axis effectively quenches I/R injury in preclinical models, there is limited efficacy of XO inhibitors in clinical trials. In this context, we considered clinical evaluation of a role for the hypoxanthine-XO axis in human I/R to be relevant. Patients undergoing renal allograft transplantation were included (n = 40) and classified based on duration of ischemia (short, intermediate, and prolonged). Purine metabolites excreted by the reperfused kidney (arteriovenous differences) were analyzed by the ultra performance liquid chromatography-tandem mass spectrometer (UPLCMS/MS) method and tissue XO activity was assessed by in situ enzymography. We confirmed progressive hypoxanthine accumulation (P < 0.006) during ischemia, using kidney transplantation as a clinical model of I/R. Yet, arteriovenous concentration differences of uric acid and in situ enzymography of XO did not indicate significant XO activity in ischemic and reperfused kidney grafts. Furthermore, we tested a putative association between hypoxanthine accumulation and renal oxidative stress by assessing renal malondialdehyde and isoprostane levels and allantoin formation during the reperfusion period. Absent release of these markers is not consistent with an association between ischemic hypoxanthine accumulation and postreperfusion oxidative stress. On basis of these data for the human kidney we hypothesize that the role for the hypoxanthine-XO axis in clinical I/R injury is less than commonly thought, and as such the data provide an explanation for the apparent limited clinical efficacy of XO inhibitors.