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To reduce treatment burden and optimise patient outcomes in diabetic macular oedema, we present 1-year results from two phase 3 trials of faricimab, a novel angiopoietin-2 and vascular endothelial growth factor-A bispecific antibody.YOSEMITE and RHINE were randomised, double-masked, non-inferiority trials across 353 sites worldwide. Adults with vision loss due to centre-involving diabetic macular oedema were randomly assigned (1:1:1) to intravitreal faricimab 6·0 mg every 8 weeks, faricimab 6·0 mg per personalised treatment interval (PTI), or aflibercept 2·0 mg every 8 weeks up to week 100. PTI dosing intervals were extended, maintained, or reduced (every 4 weeks up to every 16 weeks) based on disease activity at active dosing visits. The primary endpoint was mean change in best-corrected visual acuity at 1 year, averaged over weeks 48, 52, and 56. Efficacy analyses included the intention-to-treat population (non-inferiority margin 4 Early Treatment Diabetic Retinopathy Study [ETDRS] letters); safety analyses included patients with at least one dose of study treatment. These trials are registered with ClinicalTrials.gov (YOSEMITE NCT03622580 and RHINE NCT03622593).3247 patients were screened for eligibility in YOSEMITE (n=1532) and RHINE (n=1715). After exclusions, 940 patients were enrolled into YOSEMITE between Sept 5, 2018, and Sept 19, 2019, and 951 patients were enrolled into RHINE between Oct 9, 2018, and Sept 20, 2019. These 1891 patients were randomly assigned to faricimab every 8 weeks (YOSEMITE n=315, RHINE n=317), faricimab PTI (n=313, n=319), or aflibercept every 8 weeks (n=312, n=315). Non-inferiority for the primary endpoint was achieved with faricimab every 8 weeks (adjusted mean vs aflibercept every 8 weeks in YOSEMITE 10·7 ETDRS letters [97·52% CI 9·4 to 12·0] vs 10·9 ETDRS letters [9·6 to 12·2], difference -0·2 ETDRS letters [-2·0 to 1·6]; RHINE 11·8 ETDRS letters [10·6 to 13·0] vs 10·3 ETDRS letters [9·1 to 11·4] letters, difference 1·5 ETDRS letters [-0·1 to 3·2]) and faricimab PTI (YOSEMITE 11·6 ETDRS letters [10·3 to 12·9], difference 0·7 ETDRS letters [-1·1 to 2·5]; RHINE 10·8 ETDRS letters [9·6 to 11·9], difference 0·5 ETDRS letters [-1·1 to 2·1]). Incidence of ocular adverse events was comparable between faricimab every 8 weeks (YOSEMITE n=98 [31%], RHINE n=137 [43%]), faricimab PTI (n=106 [34%], n=119 [37%]), and aflibercept every 8 weeks (n=102 [33%], n=113 [36%]).Robust vision gains and anatomical improvements with faricimab were achieved with adjustable dosing up to every 16 weeks, demonstrating the potential for faricimab to extend the durability of treatment for patients with diabetic macular oedema.F Hoffmann-La Roche.

Faricimab is a bispecific antibody that acts through dual inhibition of both angiopoietin-2 and vascular endothelial growth factor A. We report primary results of two phase 3 trials evaluating intravitreal faricimab with extension up to every 16 weeks for neovascular age-related macular degeneration (nAMD).TENAYA and LUCERNE were randomised, double-masked, non-inferiority trials across 271 sites worldwide. Treatment-naive patients with nAMD aged 50 years or older were randomly assigned (1:1) to intravitreal faricimab 6·0 mg up to every 16 weeks, based on protocol-defined disease activity assessments at weeks 20 and 24, or aflibercept 2·0 mg every 8 weeks. Randomisation was performed through an interactive voice or web-based response system using a stratified permuted block randomisation method. Patients, investigators, those assessing outcomes, and the funder were masked to group assignments. The primary endpoint was mean change in best-corrected visual acuity (BCVA) from baseline averaged over weeks 40, 44, and 48 (prespecified non-inferiority margin of four letters), in the intention-to-treat population. Safety analyses included patients who received at least one dose of study treatment. These trials are registered with ClinicalTrials.gov (TENAYA NCT03823287 and LUCERNE NCT03823300).Across the two trials, 1329 patients were randomly assigned between Feb 19 and Nov 19, 2019 (TENAYA n=334 faricimab and n=337 aflibercept), and between March 11 and Nov 1, 2019 (LUCERNE n=331 faricimab and n=327 aflibercept). BCVA change from baseline with faricimab was non-inferior to aflibercept in both TENAYA (adjusted mean change 5·8 letters [95% CI 4·6 to 7·1] and 5·1 letters [3·9 to 6·4]; treatment difference 0·7 letters [-1·1 to 2·5]) and LUCERNE (6·6 letters [5·3 to 7·8] and 6·6 letters [5·3 to 7·8]; treatment difference 0·0 letters [-1·7 to 1·8]). Rates of ocular adverse events were comparable between faricimab and aflibercept (TENAYA n=121 [36·3%] vs n=128 [38·1%], and LUCERNE n=133 [40·2%] vs n=118 [36·2%]).Visual benefits with faricimab given at up to 16-week intervals demonstrates its potential to meaningfully extend the time between treatments with sustained efficacy, thereby reducing treatment burden in patients with nAMD.F Hoffmann-La Roche.

Xanthomonas oryzae pv. oryzae (Xoo) possesses transcription activator-like (TAL) effectors, which are important virulence factors for bacterial blight disease. Rice nucleotide-binding leucine rich repeats receptor Xa1 recognizes TAL effectors, then induces various immune responses. To overcome host defenses, Xoo has developed TAL effector variants, termed interfering TAL (iTAL) (or truncated TAL) effectors. Japanese Xoo strain T7133 overcomes Xa1-mediated immunity. In this study, we determined the whole genome sequence of Xoo T7133 and identified TAL and iTAL effectors, revealing commonalities and differences in the TAL effectors between Xoo T7133 and avirulent strain T7174. In addition, Xoo T7133 has a different type of iTAL effector from Xoo T7174, suggesting that the iTAL effector of Xoo T7133 may contribute to the suppression of Xa1-mediated immunity.

A woman in her late 40s presented with multiple abnormal shadows on high-resolution CT (HRCT), was treated with abemaciclib for recurrent right breast cancer post-surgery and chemoradiation therapy. During the 10-month chemotherapy, HRCT revealed a recurrent pattern of a partly appearing and disappearing organising pneumonia pattern without clinical symptoms. Bronchoalveolar lavage analysis revealed lymphocytosis, while transbronchial lung biopsy revealed alveolitis with epithelial cell injury. Based on the diagnosis of drug-induced pneumonitis due to abemaciclib, the discontinuation of abemaciclib and administration of prednisolone were effective. Abnormal shadow on HRCT disappeared gradually, while elevated Krebs von den Lungen (KL)-6 and surfactant protein (SP)-D levels were restored to normal range. This is the first case report of abemaciclib-induced pneumonitis with histology findings. Since the severity of abemaciclib-induced pneumonitis ranges from mild to fatal, regular monitoring of pneumonitis with radiography, HRCT, and measurement of KL-6 and SP-D levels should be considered.

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.

SummaryPlant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses and the hypersensitive response by recognizing pathogen effectors. Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo). The molecular mechanisms controlling the recognition of TAL effectors by Xa1 and the subsequent induction of immunity remain poorly understood.Xa1 interacts in the nucleus with two TAL effectors via the BED domain. We identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6 as an interactor with Xa1, and found that it also interacts with the TAL effectors.Overexpression of OsERF101 exhibited an enhanced resistance to an incompatible Xoo strain only in the presence of Xa1, indicating that OsERF101 functions as a positive regulator of Xa1-mediated immunity. Unexpectedly, oserf101 mutants also showed enhanced Xa1-dependent resistance, but in a different manner from the overexpressing plants. This result revealed an additional Xa1-mediated immune pathway that is negatively regulated by OsERF101. Furthermore, OsERF101 directly interacted with the TAL effectors.Our results show that OsERF101 regulates the recognition of TAL effectors and the Xa1-mediated activation of the immune response. These data provide new insights into the molecular mechanism of NLR-mediated immunity in plants.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses by recognizing pathogen effectors. The rice gene Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo). Our goal is to elucidate the molecular mechanisms controlling the induction of immunity by Xa1. We used yeast two-hybrid assays to screen for host factors that interact with Xa1 and identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6. Molecular complementation assays were used to confirm the interactions among Xa1, OsERF101, and two TAL effectors. We created OsERF101-overexpressing and knockout mutant lines in rice and identified genes differentially regulated in these lines, many of which are predicted to be involved in regulation of response to stimulus. Xa1 interacts in the nucleus with the TAL effectors and OsERF101 via the BED domain. Unexpectedly, both the overexpression and knockout lines of OsERF101 displayed Xa1-dependent, enhanced resistance to an incompatible Xoo strain. Different sets of genes were up- or down-regulated in the overexpression and knockout lines. Our results indicate that OsERF101 regulates the recognition of TAL effectors by Xa1, and functions as a positive regulator of Xa1-mediated immunity. Further, an additional Xa1-mediated immune pathway is negatively regulated by OsERF101., イネが病原菌の感染力の源を検出して免疫を誘導する仕組みを解明 --病気に強い植物の開発に期待--. 京都大学プレスリリース. 2022-09-07.

Cigarette smoking in patients with asthma leads to poor symptom control. As patients who are current smokers have been excluded from enrollment in many clinical trials on asthma, there are few reports on the treatment in current smokers with asthma. In this study, we aimed to assess how respiratory physicians manage asthma in current smokers in Japan.Respiratory physicians in 16 Japanese hospitals answered a questionnaire on treatment for patients with asthma between December 2014 and February 2015. Medical records were reviewed for 1756 patients with asthma.The mean patient age was 61.1 years, and 62.9% of the patients were female. A total of 102 patients (5.8%) were current smokers, and 546 patients (31.1%) were former smokers. Long-acting muscarinic antagonists (LAMA) were prescribed more frequently for current smokers with asthma than for former smokers and never smokers with asthma (10.8% vs 4.6%, p = 0.01, 10.8% vs 3.8%, p0.01). In contrast, macrolides were prescribed more frequently for former smokers and never smokers with asthma than for current smokers with asthma (7.7% vs 1.0%, p = 0.01, 6.4% vs 1.0%, p = 0.03). Triple therapy, i.e., inhaled corticosteroids, long-acting beta agonists, and LAMA concomitantly, was prescribed for current smokers with asthma more frequently than for former smokers and never smokers with asthma (9.8% vs 4.0%, p = 0.01, 9.8% vs 3.3%, p0.01).According to this survey, current smokers with asthma received more intensive therapy, including LAMA, than did former smokers with asthma.

The rice receptor-like cytoplasmic kinase 185 (OsRLCK185) interacts with the chitin receptor complex OsCERK1/CEBiP and positively regulates chitin-induced immune responses including MAP kinase activation, ROS production and defense gene expression. To elucidate the regulatory mechanisms of OsRLCK185-mediated immunity, we searched for interactors of OsRLCK185. OsDRE2a, rice homologs of the yeast Dre2 protein, were identified as novel interactors of OsRLCK185. OsDRE2a interacted with OsRLCK185 at plasma membrane. The conserved cysteine residues in CIAPIN1 domain of OsDRE2a were essential for tight interaction of OsRLCK185. OsDRE2a was phosphorylated by OsRLCK185. The expression of OsDRE2a and OsDRE2b was induced after chitin treatment. Reduction of OsDRE2a and OsDRE2b mRNA levels by RNA interference resulted in the decreased chitin-induced ROS production. Thus, it is likely that OsDRE2 regulates OsRLCK185-mediated immune responses.

A 52-year-old woman presented to a clinic in late August with exacerbated fatigue and dyspnea on exertion for several months. Then, she was referred and admitted to our hospital in late September. Her chest CT showed bilateral diffuse centrilobular micronodules. In her detailed clinical history, she had kept budgerigars indoors for 15 years. These findings suggested she had a bird-related hypersensitivity pneumonitis (BRHP). By a site environmental investigation, 40 budgerigars were kept in a single breeding room and there were large amounts of droppings on the floor. Serum specific antibody for bird antigens and an environmental provocation test were positive. Bronchoalveolar lavage fluid showed lymphocytosis and a low CD4/CD8 ratio. Trans-bronchial lung biopsy showed lymphocytic infiltration of the alveolar wall and interlobular septa. After antigen avoidance as hospitalization, her symptoms and abnormal shadow improved. From these results, the patient was diagnosed as an acute BRHP.BRHP often presents a chronic onset. This case was diagnosed as an acute type despite the 15-years of budgerigars breeding. Increased exposure of antigens due to lack of cleaning after several days' antigen avoidance was suspected with one of the causes of acute onset.

The U-box type ubiquitin ligase PUB44 is targeted by the Xanthomonas oryzae XopP effector and positively regulates pattern-triggered immunity in rice. Here we identified PBI1, a protein that interacts with PUB44. Crystal structure analysis indicated that PBI1 forms a four-helix bundle structure. PBI1 also interacts with WRKY45, a key transcriptional activator of rice immunity, and negatively regulates its activity. PBI1 is degraded during the chitin response, and this is suppressed by silencing of PUB44 or expression of XopP, indicating that PBI1 degradation depends on PUB44. These data suggest that PBI1 suppresses WRKY45 activity when cells are in the unelicited state, and during chitin signaling, PUB44-mediated degradation of PBI1 leads to activation of WRKY45. In addition, phosphorylation of WRKY45 by MAP kinases releases WRKY45 from the PBI1-mediated inhibitory effect. These results demonstrate that chitin-induced activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation and PUB44-mediated PBI1 degradation.

We investigated the clinical course of individuals with 2019 novel coronavirus disease (COVID-19) who were transferred from the Diamond Princess cruise ship to 12 local hospitals. The conditions and clinical courses of patients with pneumonia were compared with those of patients without pneumonia. Among 70 patients (median age: 67 years) analyzed, the major symptoms were fever (64.3%), cough (54.3%), and general fatigue (24.3%). Forty-three patients (61.4%) had pneumonia. Higher body temperature, heart rate, and respiratory rate as well as higher of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and C-reactive protein (CRP) levels and lower serum albumin level and lymphocyte count were associated with the presence of pneumonia. Ground-glass opacity was found in 97.7% of the patients with pneumonia. Patients were administered neuraminidase inhibitors (20%), lopinavir/ritonavir (32.9%), and ciclesonide inhalation (11.4%). Mechanical ventilation and veno-venous extracorporeal membrane oxygenation was performed on 14 (20%) and 2 (2.9%) patients, respectively; two patients died. The median duration of intubation was 12 days. The patients with COVID-19 transferred to local hospitals during the outbreak had severe conditions and needed close monitoring. The severity of COVID-19 depends on the presence of pneumonia. High serum LDH, AST and CRP levels and low serum albumin level and lymphocyte count were found to be predictors of pneumonia. It was challenging for local hospitals to admit and treat these patients during the outbreak of COVID-19. Assessment of severity was crucial to manage a large number of patients.

Plants are constantly exposed to pathogens in their immediate environment. Plants sense the invasion of pathogens by recognizing the components including peptide fragments derived from pathogens, known as pathogen-associated molecular patterns (PAMPs). Plants also produce immunogenic peptides called phytocytokines that regulate immune responses. These molecules are recognized by pattern recognition receptors (PRRs) at plasma membrane. Activated PRRs induce a variety of immune responses including production of reactive oxygen species (ROS), induction of Ca2+ influx and activation of mitogen activated protein kinases (MAPKs). Pattern-triggered immunity (PTI) wards off microbes and pests. In this review, we summarize recent our advances in understanding how the peptide fragments are generated and perceived by plant PRRs at cell surface, and the activated PRRs transduce the downstream immune signaling.

Perception of microbe-associated molecular patterns (MAMPs) including chitin by pattern recognition receptors (PRRs) rapidly induces activation of mitogen-activated protein kinase (MAPK) cascades. However, how PRRs transmit immune signals to the MAPK cascade is largely unknown. Recently, Arabidopsis receptor-like cytoplasmic kinase PBL27 has been reported to activate MAPKs through phosphorylation of AtMAPKKK5 in the chitin signaling pathway. In this study, we found that OsRLCK185, a rice ortholog of PBL27, regulates chitin-induced MAPK activation in a similar fashion to PBL27 in rice. Upon chitin perception, OsRLCK185 is phosphorylated by OsCERK1, a component of the chitin receptor complex. OsRLCK185 interacted with OsMAPKKK11 and OsMAPKKK18, rice orthologs of AtMAPKKK5, in yeast two-hybrid assays. Silencing of both OsMAPKKK11 and OsMAPKKK18 significantly reduced chitin-induced activation of OsMPK3 and OsMPK6. Expression levels of OsMAPKKK18 were much higher than that of OsMAPKKK11 in rice cells, which was consistent with the fact that the Osmapkkk11 single mutation did not affect MAPK activation. This result suggested that OsMAPKKK18 plays a more important role than OsMAPKKK11 in the chitin-induced activation of OsMPK3 and OsMPK6. The bimolecular fluorescence complementation (BiFC) experiment indicated that OsRLCK185 interacted with OsMAPKKK18 at the plasma membrane in planta. In vitro phosphorylation experiments showed that OsRLCK185 directly phosphorylates OsMAPKKK18. Furthermore, OsMAPKKK18 interacted with the MAPKK OsMKK4, the upstream component of OsMPK3/6. These results suggested that OsRLCK185 connects the chitin receptor to the MAPK cascade consisting of OsMAPKKK18-OsMKK4-OsMPK3/6. Our data revealed that chitin-induced MAPK activation in rice and Arabidopsis is regulated by common homologous elements.

Introduction: The prevalence of asthma in the elderly has been increasing due to the aging of the global population. Appropriate treatment for asthma in the elderly is now a major issue, as the disease is often fatal and incurs high medical costs in the elderly population. There have been few recent reports on asthma treatment for the elderly in the real-world clinical settings. Objectives: This study was performed to assess how respiratory physicians manage asthma in elderly patients in clinical settings in Japan. Methods: Respiratory physicians in 16 Japanese hospitals responded to a questionnaire survey on asthma and COPD treatment between December 2014 and February 2015. The analysis was performed using data collected from medical records on 2041 asthma patients. Results: The mean patient age was 61.2 years, and 36.6% of the patients were men. In the breakdown by age, 1018 (49.9%) of the patients were elderly (≥ 65 years). Compared to the non-elderly, the elderly patients had a significantly lower BMI, greater smoking history in pack-years, and poorer pulmonary function. There were no significant differences between the elderly and non-elderly in the prescription rates of common medications such as inhaled corticosteroids, long-acting beta-agonists (LABA) administered via inhaler or transdermal patch, long-acting muscarinic antagonists, or leukotriene receptor antagonists. In contrast, mucoactive drugs (18.7% vs. 12.3%, P P P = 0.02). Conclusions: According to this survey, the prescription rates of mucoactive drugs, macrolides, and transdermal LABA were significantly higher in the elderly than in the non-elderly.

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola . PBL27 phosphorylates MAPKKK5 in vitro , which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo . Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants. ![][1] Chitin receptor CERK1 transmits immune signals to the intracellular MAPK cascade in plants. This occurs via phosphorylation of MAPKKK5 by the CERK1‐associated kinase PBL27, providing a missing link between pathogen perception and signaling output. [1]: /embed/graphic-1.gif

Background Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. Results We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images. Conclusions Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein–protein interactions for other FRET sensors in various plant cells.

A 47-year old man presented to our hospital with a 6-month history of malaise, cough and dyspnea on exertion. Laboratory testing revealed the severe hyponatremia. A chest X-ray showed bilateral diffuse micronodules. Anti-Trichosporon asahii antibody and environmental provocation test were positive. Bronchoalveolar lavage fluid showed lymphocytosis and low CD4/8 ratio. The specimens obtained by transbronchial lung biopsy revealed alveolitis. Based on these findings, the patient was diagnosed as having summer-type hypersensitivity pneumonitis (SHP). The patient was treated with antigen avoidance and oral corticosteroid. The hyponatremia caused by syndrome of inappropriate secretion of antidiuretic hormone (SIADH) was treated with normal saline and water restriction. Serum sodium level was improved with treatment of SHP, which suggested the relevance between SHP and SIADH.

Although the pronator teres muscle, a major dynamic stabilizer of elbow valgus stress during throwing, frequently presents stiffness, its relationship with elbow injuries in youth baseball players is unknown. This study investigated the relationship between the elasticity of the pronator teres muscle and elbow injuries in youth baseball players.The strain ratio (SR) of 15 individuals with osteochondritis dissecans of the humeral capitellum (OCD group), 67 individuals with medial epicondylar fragmentation (medial injury group), and 115 healthy individuals (control group) was measured as the index of the elasticity of the pronator teres muscle using ultrasound strain elastography. In addition, the forearm and glenohumeral joint rotation range of motion was measured.The SR of the throwing arm was significantly higher in the OCD and medial injury groups than in the control group (both P .001). In the OCD group, the SR was significantly higher in the throwing arm than in the nonthrowing arm (P .001), whereas in the medial injury group, there was no significant difference between both arms. The glenohumeral joint external rotation range of motion of the throwing arm was moderately negatively correlated with the SR (r = -0.478, P .001).Stiffness of the pronator teres muscle was exhibited only in the throwing arm of individuals with OCD and in both arms in individuals with medial elbow injury. These findings may contribute to an accurate evaluation and prevention of elbow injuries in youth baseball players.

Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in rice cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts withRaichu-OsRac1and acquisition of FRET images. Our protocol should be widely adaptable for monitoring activation for other plant small GTPases and for other FRET sensors in various plant cells.

Rice blast and bacterial blight, two important rice diseases, are caused by infection with a fungal pathogen Magnaporthe oryzae and a bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), respectively. Recent studies on the interaction between rice and these pathogens provided important knowledges of the molecular mechanisms of rice immune responses such as receptor-mediated pathogen recognition, host immune signaling, and pathogen effector-mediated susceptibility. So far, many disease resistance (R) genes have been genetically identified based upon disease resistance traits against M. oryzae and Xoo. Most rice blast R genes isolated to date encode pathogen recognition receptors. In contrast, the majority of bacterial blight R genes are involved in transcriptional regulation of host resistance or susceptibility factors. Genetic and biochemical studies of rice immune signaling have identified important immune factors including OsRac1, OsRLCK185, and WRKY45. Identification of rice factors that interact with OsRac1, OsRLCK185, and WRKY45 revealed the molecular mechanisms of a variety of immune responses, including the expression of defense-related genes, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK), and lignification.

JAEA is fabricating the toroidal field insert coil (TFIC) in cooperation with Hitachi, Ltd. This solenoidal coil has about 50 m of ITER TF conductor wound in a 1.44-m diameter. In preparation for fabricating the TFIC, fabrication trials of windings, removal of Cr plating of the cable and welding of the terminal sleeve were performed for process qualification. The winding trials were accomplished without breaking any of the superconducting strands. In the trials to remove the Cr plating from the cable, HCl-soaked unwoven cloth was used, and the surface of a strand selected from the cable was confirmed by magnification to be free of Cr plating. In trials to fabricate termination, using electron beam welding (of OFHC copper and SS316LN) and fillet-welding (of SS316LN and SS316LN using JK2LB weld wire), tensile tests of the welds were conducted at room temperature and 4 K, and ultimate tensile strength values equivalent to that of the base metal of the weld were obtained. Also these welds passed test items of JIS Z3040 code, so the weld procedures were qualified. During fillet-welding the maximum temperature of the cable under the weld was 120 °C, not high enough to damage the cable. From the results obtained, the main processes involved in TFIC fabrication were established and qualified.

We report two family members, a 64-year-old woman (patient 1) and her 37-year-old son (patient 2) diagnosed with summer-type hypersensitivity pneumonitis (SHP). Both patients had high serum titers of anti-Trichosporon asahii antibody. The patients lived in the same house and worked in the same barbershop. Patient 1 was diagnosed with SHP in the summer, and she reacted positively to the provocation test at the work place, but not in the house. Patient 2 was diagnosed with SHP in the winter. Generally, SHP develops and is diagnosed in the summer. The home environment is responsible for most cases of familial SHP. Therefore, our cases of familial SHP are unusual and may suggest that the clinical characteristics of SHP have changed, due to alterations in social and environmental conditions.

Suspension-cultured cells respond sensitively to a number of stimuli including pathogen-derived molecules. Therefore, they are used in a simple, single-cell model system in order to gain insights into immune signaling pathways in rice. Here we describe protocols for studying chitin-induced MAPK activation and ROS generation in rice suspension-cultured cells.

Suspension-cultured cells respond sensitively to a number of stimuli including pathogen-derived molecules. Therefore, they are used in a simple, single-cell model system in order to gain insights into immune signaling pathways in rice. Here we describe protocols for studying chitin-induced MAPK activation and ROS generation in rice suspension-cultured cells.

This research was originally published in Journal of Biological Chemistry. Ken-ichi Kosami, Izuru Ohki, Minoru Nagano, Kyoko Furuita, Toshihiko Sugiki, Yoji Kawano, Tsutomu Kawasaki, Toshimichi Fujiwara, Atsushi Nakagawa, Ko Shimamoto and Chojiro Kojima. The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase. Journal of Biological Chemistry. 2014; 289, 28569-28578. © the American Society for Biochemistry and Molecular Biology., Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5´-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate theROSproduction in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr^39 and Asp^45 substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

Under the International Thermonuclear Experimental Reactor (ITER) project, the Japan Atomic Energy Agency (JAEA) is procuring all of the Nb3Sn conductors for the Central Solenoid (CS). The CS consists of six vertically stacked modules. The height and outer diameter of the CS are approximately 13 m and 4 m, respectively. The CS has a circular five stage cable. All of approximately 43 km of Nb3Sn CS cables will be manufactured in Japan. Before mass-production start, the jacketed cable conductors should be tested in the SULTAN facility in Switzerland to confirm their superconducting performance. The original cabling design had relatively long twist pitches and is referred to as the normal twist pitch (NTP) conductor. The NTP conductor test results revealed decreasing the current sharing temperature (Tcs) with increasing number of electro-magnetic (EM) load cycles. Therefore, a short twist pitch (STP) design was proposed and the STP conductors were also tested. The STP conductor results showed that the Tcs is stable during EM cyclic load tests. Because the conductors with STP have a smaller void fraction in the cable area than those with NTP, a higher compaction ratio during cabling is required and the possibility of damage on strands increases. The STP cable technology was developed in collaboration among Japanese cabling suppliers and JAEA. Several key technologies will be described in this paper.

Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.

The Os01T0156300 protein fromOryza sativahas been classified into the domain of unknown function (DUF) family DUF1110. DUF1110 family members exist in monocotyledons but not in dicotyledons, and share no sequence identity with proteins for which structures have been reported. In this study, the Os01T0156300 protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.84 Å resolution. The crystal belonged to space groupP21, with unit-cell parametersa= 89.9,b= 89.8,c= 107.1 Å, β = 106.6°. The asymmetric unit was estimated to contain 6–11 molecules.

In animals, major classes of Rho guanine nucleotide exchange factors (GEFs) possess a Dbl (diffuse B-cell lymphoma)- homology (DH) domain that functions as a GEF-catalytic domain. However, no GEFs with the DH domain had been identified in plants. Recently, we found that the rice homolog of human SWAP70, Oryza sative (Os) SWAP70, containing the DH domain, exhibited GEF activity toward the rice Rho GTPase OsRac1, and regulates chitin-induced production of reactive oxygen species and defense gene expression in rice. ( 1) Arabidopsis contains a single SWAP70 gene. A T-DNA insertion mutant of Arabidopsis SWAP70 was morphologically wild type. Measurement of in planta growth of Pseudomonas syringae DC3000 hrcC, a mutant incapable of type III effector delivery, revealed enhanced growth of the pathogen in the atswap70 mutant, indicating that AtSWAP70 is required for PAMP-triggered immunity. In addition, the atswap70 mutation reduced the RPM1-mediated hypersensitive response. These results suggested that AtSWAP70 plays a role in both PAMP- and effector-triggered immunity in Arabidopsis.

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.

SummaryThe nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

Plant-specifi c Rac/Rop small GTPases function as molecular switches for numerous signal transduction events, including defense responses. To understand the function of each of the seven Rac/Rop family members in rice, we studied the tissue-specifi c expression patterns of Rac/Rop genes by semi-quantitative reverse transcription–PCR (RT–PCR), and also Rac/Rop subcellular localization using green fl uorescent protein (GFP) fusion proteins in transient expression systems. We also investigated the roles of these genes in disease resistance by testing single Rac/Rop -RNAi (RNA interference) plants against the rice blast pathogen Magnaporthe grisea . Our studies show that expression of OsRac2 , 6 and 7 is very low in leaf blades, and reveal a strong correlation between the number of lysine and/or arginine (KR) residues in the polybasic region of Rac/Rop GTPases and their subcellular distribution in vivo. Infection assays showed that OsRac1 is a positive regulator of blast resistance, confi rming previous observations, whereas OsRac4 and OsRac5 are negative regulators of blast resistance. OsRac6 may make minor contributions to disease resistance, while OsRac3 and OsRac7 are probably not involved in defense. Therefore, our study suggests that the rice Rac/Rop family plays multiple roles in diverse cellular activities and has both positive and negative functions in disease resistance.