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Abstract An inhibitor of plasminogen activator inhibitor (PAI)-1, TM5614, inhibited thrombosis, inflammation, and fibrosis in several experimental mouse models. To evaluate the efficacy and safety of TM5614 in human COVID-19 pneumonia, phase IIa and IIb trials were conducted. In an open-label, single-arm trial, 26 Japanese COVID-19 patients with mild to moderate pneumonia were treated with 120–180 mg of TM5614 daily, and all were discharged without any notable side effects. Then, a randomized, double-blind, placebo-controlled trial was conducted in Japanese COVID-19 patients with mild to moderate pneumonia. The number of study participants was set to be 50 in each arm. Even after extension of the enrollment period, the number of study participants did not reach the initially intended sample size, and 75 patients were enrolled in the study. The total oxygenation scale from Day 1 to Day 14 as the primary endpoint was 1.5 in the TM5614 group vs 4.0 in the placebo group (p = 0.22), and the number of days of oxygen administration required as the secondary endpoint was 2.0 days in the TM5614 group vs 3.5 days in the placebo group (p = 0.34). Further studies will be necessary to verify the efficacy of PAI-1 inhibition for the treatment of COVID-19 pneumonia. Clinical trial registration: Two studies were conducted: a prospective, multicenter, open-label phase II study at https://jrct.niph.go.jp (jRCT2021200018) (First registration date 18/08/2020) and a prospective, multicenter, randomized, double-blind, placebo-controlled, phase II study at https://jrct.niph.go.jp (jRCT2021210006) (First registration date 28/05/2021).

The U-box type ubiquitin ligase PUB44 positively regulates pattern-triggered immunity in rice. Here the authors identify a PUB44 substrate whose degradation is required for activation of the WRKY45 transcription factor upon immune elicitation.

Abstract 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.

In Arabidopsis, fungal chitin is recognized as a pathogen-associated molecular pattern (PAMP) by the chitin receptor complex containing the lysin-motif (LysM) receptor-like kinases CERK1 and LYK5. Upon the perception of chitin, CERK1 phosphorylates the receptor-like cytoplasmic kinase, PBL27, which activates the intracellular mitogen-activated protein kinase (MAPK) cascade. However, the mechanisms by which the CERK1-PBL27 complex is regulated remain largely unknown. We identified ubiquitin ligase PUB12 as a component of the PBL27 complex using co-immunoprecipitation and mass spectrometry. However, PUB12 did not interact directly with PBL27. Instead, the ARM domains of PUB12 and its paralog PUB13 interacted with the intracellular domain of CERK1 in a manner that was dependent on its autophosphorylation, suggesting that the phosphorylation-based auto-activation of CERK1 may be required for its interaction with PUB12. The co-expression of PUB12 in Nicotiana benthamiana reduced the accumulation of CERK1. The pub12 pub13 mutant exhibited enhanced chitin-induced immune responses such as ROS production, MAPK activation, and callose deposition. These results suggest that PUB12 and PUB13 are involved in the negative regulation of the chitin receptor complex, which may contribute to the transient desensitization of chitin-induced responses.

Hiroko Sonoyama1, Kaoru Araki-Sasaki1, Shigeyasu Kazama1, Tsutomu Kawasaki1, Hidenao Ideta1, Atsuko Sunada2, Seishi Asari2, Yoshitsugu Inoue3, Kozaburo Hayashi41Ideta Eye Hospital, Kumamoto, Kumamoto, Japan; 2Department of Laboratory for Clinical Investigation, Osaka University Hospital, Suita, Osaka, Japan; 3Division of Ophthalmology and Visual Science, Department of Medicine of Sensory and Motor Organ, Tottori University, Yonago, Tottori, Japan; 4Immunology and Virology Section Lab, Immunology, NEI, NIH, Bethesda, MD, USAAbstract: Clinical findings and treatment of keratomycosis caused by Beauveria bassiana, an entomopathogenic filamentous fungus, are described for an 80-year-old woman, who was referred to the hospital for ocular pain and redness on the 9th day after an ocular injury caused by the frame of her glasses. She had a long history of recurrent diabetic iritis and continuously used topical antibiotics and corticosteroids. At her first visit, a slit-lamp examination indicated a corneal ulcer confined within the superficial stromal layer, along with a slight infiltration and edema. Only a very few inflammatory cells were seen in the anterior chamber. Direct microscopic examination of corneal scrapings revealed septate fungal hyphae with zig-zag rachis and budding that was subsequently identified as B. bassiana by slide culture. Topical voriconazole with miconazole, pimaricin and oral itraconazole were effective and the lesion disappeared leaving only a mild scar at 2 months. The sensitivity of B. bassiana to various antimycotic agents was confirmed by broth microdilution, agar dilution with the Clinical Laboratory Standard Institute standard, and a disk method using topically applied concentrations. B. bassiana, which exhibits a characteristic appearance in smears and causes superficial keratomycosis, is sensitive to voriconazole with miconazole, pimaricin, and itraconazole.Keywords: Beauveria bassiana, keratomycosis, filamentous fungus, voriconazole, corneal infection

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.

Kaoru Araki-Sasaki1, Hiroko Sonoyama1, Tsutomu Kawasaki1, Nariyasu Kazama1, Hidenao Ideta1, Yoshitsugu Inoue21Ideta Eye Hospital, Kumamoto City, Kumamoto, Japan; 2Department of Ophthalmology, Tottori University, Koyama-Minami, Tottori, Japan Abstract: The paper reports on Candida albicans ocular infection modified by steroid eye drops. A 74-year-old male complained of conjunctival injection and pain in his right eye three months after pterygium and cataract surgery. After treatment with antibiotics and steroid eye drops for three days, he was referred to our hospital. Clear localized corneal endothelial plaque with injection of ciliary body was observed. No erosion of the corneal epithelium, or infiltration of stromal edema was observed, suggesting that the pathological organism derived from the intracameral region. Because ocular infection was suspected, steroid eye drops were stopped, which led immediately to typical infectious keratitis in the pathological region, with epithelial erosion, fluffy abscess, stromal infiltration, and edema. For diagnostic purposes, the plaque was surgically removed with forceps and the anterior chamber was irrigated with antibiotics. The smear and culture examination from the plaque revealed C. albicans surrounded by neutrophils. However, aqueous fluid and fibrous tissue after gonio procedure contained no mycotic organisms. Topical fluconazole, micafungin, and pimaricin with oral itraconazole (150 mg/day) were effective. Special attention is needed when prescribing steroid eye drops to treat corneal disease especially postoperatively. Diagnosing infectious keratitis is sometimes difficult because of modification by some factors, such as postoperative conditions, scarring, and drug-induced masking. Here, we report on mycotic keratitis modified by postoperative steroid administration. Keywords: Candida albicans, cataract surgery, steroid, mycotic keratitis

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

OsRac1, a member of the Rac/Rop GTPase family, plays important roles as a molecular switch in rice innate immunity, and the active form of OsRac1 functions in the plasma membrane (PM). To study the precise localization of OsRac1 in the PM and its possible association with other signaling components, we performed proteomic analysis of DRMs (detergent-resistant membranes) isolated from rice suspension-cultured cells transformed with myc-tagged constitutively active (CA) OsRac1. DRMs are regions of the PM that are insoluble after Triton X-100 treatment under cold conditions and are thought to be involved in various signaling processes in animal, yeast and plant cells. We identified 192 proteins in DRMs that included receptor-like kinases (RLKs) such as Xa21, nucleotide-binding leucine-rich repeat (NB-LRR)-type disease resistance proteins, a glycosylphosphatidylinositol (GPI)-anchored protein, syntaxin, NADPH oxidase, a WD-40 repeat family protein and various GTP-binding proteins. Many of these proteins have been previously identified in the DRMs isolated from other plant species, and animal and yeast cells, validating the methods used in our study. To examine the possible association of DRMs and OsRac1-mediated innate immunity, we used rice suspension-cultured cells transformed with myc-tagged wild-type (WT) OsRac1 and found that OsRac1 and RACK1A, an effector of OsRac1, shifted to the DRMs after chitin elicitor treatment. These results suggest that OsRac1-mediated innate immunity is associated with DRMs in the PM. [ABSTRACT FROM AUTHOR]