The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific ‘‘avirulent’’ pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NBLRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector

Single-Step Selection of Bivalent Aptamers Validated by Comparison with SELEX Using High-Throughput Sequencing

The identification of nucleic acid aptamers would be advanced if they could be obtained after fewer rounds of selection and amplification. In this paper the identification of bivalent aptamers for thrombin by SELEX and single-step selection are compared using next generation sequencing and motif finding informatics. Results show that similar aptamers are identified by both methods. This is significant because it shows that next generation sequencing and motif finding informatics have the potential to simplify the selection of aptamers by avoiding multiple rounds of enzymatic transcription and amplification

Filgotinib (GLPG0634/GS-6034), an oral JAK1 selective inhibitor, is effective in combination with methotrexate (MTX) in patients with active rheumatoid arthritis and insufficient response to MTX: results from a randomised, dose-finding study

Objectives: To evaluate the efficacy and safety of different doses and regimens of filgotinib, an oral JAK1 inhibitor, as add-on treatment to methotrexate (MTX) in patients with active rheumatoid arthritis (RA) and inadequate response to MTX. Methods: In this 24-week phase 2b study, patients with moderate-to-severe active RA receiving a stable dose of MTX were randomised (1:1:1:1:1:1:1) to receive placebo or 50 mg, 100 mg or 200 mg filgotinib, administered q.d. or b.i.d. Primary endpoint was the percentage of patients achieving a Week 12 ACR20 response. Results: Overall, 594 patients were randomised and treated. At Week 12 significantly more patients receiving filgotinib 100 mg q.d. or 200 mg daily (both regimens) achieved an ACR20 response versus placebo. For other key endpoints at Week 12 (ACR50, ACR-N, DAS28[CRP], CDAI, SDAI and HAQ-DI), differences in favour of 100 mg or 200 mg filgotinib daily were seen versus placebo; responses were maintained or improved through to Week 24. Rapid onset of action and dose-dependent responses were observed for most efficacy endpoints and were associated with an increased haemoglobin concentration. No significant differences between q.d. and b.i.d. regimens were seen. TEAE event rates were similar in placebo and filgotinib groups. Serious infections occurred in 1 and 5 patients in the placebo and filgotinib groups, respectively. No tuberculosis or opportunistic infections were reported. Conclusions: Filgotinib as add-on to MTX improved the signs and symptoms of active RA over 24 weeks, and was associated with a rapid onset of action. Filgotinib was generally well tolerated

A Grafting Strategy for the Design of Improved G-Quadruplex Aptamers and High-Activity DNAzymes

Nucleic acid aptamers are generally obtained by in vitro selection. Some have G-rich consensus sequences with ability to fold into the four-stranded structures known as G-quadruplexes. A few G-quadruplex aptamers have proven to bind hemin to form a new class of DNAzyme with the peroxidase-like activity, which can be significantly promoted by appending an appropriate base-pairing duplex onto the G-quadruplex structures of aptamers. Knowing the structural role of base pairing, here we introduce a novel grafting strategy for the design of improved G-quadruplex aptamers and high-activity DNAzymes. To demonstrate this strategy, three existing G-quadruplex aptamers are chosen as the first generation. A base-pairing DNA duplex is grafted onto the G-quadruplex motif of the first generation aptamers. Consequently, three new aptamers with the quadruplex/duplex DNA structures are produced as the second generation. The hemin-binding affinities and DNAzyme functions of the second generation aptamers are characterized and compared with the first generation. The results indicate three G-quadruplex aptamers obtained by the grafting strategy have more excellent properties than the corresponding original aptamers. Our findings suggest that, if the structures and functions of existing aptamers are thoroughly known, the grafting strategy can be facilely utilized to improve the aptamer properties and thereby producing better next-generation aptamers. This provides a simple but effective approach to the design of nucleic acid aptamers and DNAzymes

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance

Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344–733) is sufficient to bind TFT1. Removal of amino acids 605–733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis

A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption

Filgotinib versus placebo or adalimumab in patients with rheumatoid arthritis and inadequate response to methotrexate: a phase III randomised clinical trial

Objective To evaluate the efficacy and safety of the Janus kinase-1-preferential inhibitor filgotinib versus placebo or tumour necrosis factor-alpha inhibitor therapy in patients with active rheumatoid arthritis (RA) despite ongoing treatment with methotrexate (MTX).Methods This 52-week, multicentre, double-blind, placebo-controlled and active-controlled phase III trial evaluated once-daily oral filgotinib in patients with RA randomised 3:3:2:3 to filgotinib 200 mg (FIL200) or filgotinib 100 mg (FIL100), subcutaneous adalimumab 40 mg biweekly, or placebo (through week 24), all with stable weekly background MTX. The primary endpoint was the proportion of patients achieving 20% improvement in American College of Rheumatology criteria (ACR20) at week 12. Additional efficacy outcomes were assessed sequentially. Safety was assessed from adverse events and laboratory abnormalities.Results The proportion of patients (n=1755 randomised and treated) achieving ACR20 at week 12 was significantly higher for FIL200 (76.6%) and FIL100 (69.8%) versus placebo (49.9%; treatment difference (95% CI), 26.7% (20.6% to 32.8%) and 19.9% (13.6% to 26.2%), respectively; both p<0.001). Filgotinib was superior to placebo in key secondary endpoints assessing RA signs and symptoms, physical function and structural damage. FIL200 was non-inferior to adalimumab in terms of Disease Activity Score in 28 joints with C reactive protein <= 3.2 at week 12 (p<0.001); FIL100 did not achieve non-inferiority. Adverse events and laboratory abnormalities were comparable among active treatment arms.Conclusions Filgotinib improved RA signs and symptoms, improved physical function, inhibited radiographic progression and was well tolerated in patients with RA with inadequate response to MTX. FIL200 was non-inferior to adalimumab.Pathophysiology and treatment of rheumatic disease