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All aspects of plant physiology are influenced by temperature. Changes in environmental temperature alter the temperatures of plant tissues and cells, which then affect various cellular activities, such as gene expression, protein stability and enzyme activities. In turn, changes in cellular activities, which are associated with either exothermic or endothermic reactions, can change the local temperature in cells and tissues. In the past 10 years, a number of fluorescent probes that detect temperature and enable intracellular temperature imaging have been reported. Intracellular temperature imaging has revealed that there is a temperature difference >1°C inside cells and that the treatment of cells with mitochondrial uncoupler or ionomycin can cause more than a 1°C intracellular temperature increase in mammalian cultured cells. Thermogenesis mechanisms in brown adipocytes have been revealed with the aid of intracellular temperature imaging. While there have been no reports on plant intracellular temperature imaging thus far, intracellular temperature imaging is expected to provide a new way to analyze the mechanisms underlying the various activities of plant cells. In this review, I will first summarize the recent progress in the development of fluorescent thermometers and their biological applications. I will then discuss the selection of fluorescent thermometers and experimental setup for the adaptation of intracellular temperature imaging to plant cells. Finally, possible applications of intracellular temperature imaging to investigate plant cell functions will be discussed.

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in theArabidopsis thalianagenome are grouped into four subclasses, and subclass I ADFs, ADF1–4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreduplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through a microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in bothadf4and transgenic lines where expression of subclass IADFs are downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0.A. thalianapossesses eightACTINgenes, among whichACT2, -7, and-8are expressed in vegetative organs. The chromocenter size inact7, but not in theact2/8double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed inadf4andADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat (NLR) genes, which are involved in effector-triggered plant immunity, was reduced inadf4andADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACTIN regulate nuclear organization and gene expression is discussed.

Purpose: To evaluate the clinical improvement and safety of prolonged treatment of vernal (VKC) and atopic keratoconjunctivitis (AKC) using topical tacrolimus. Methods: We included 36 eyes of 36 patients who had VKC and AKC and were treated with topical tacrolimus ophthalmic suspension (0.1%) for 24 months. The demographic data of the enrolled patients were collected from their medical files. Clinical scores, remission rates, number of relapses, concomitant use of steroids, and refractory indices were assessed. Clinical outcomes were determined using papillae–limbus–cornea (PLC) scores and 5-5-5 exacerbation grading scale scores. Clinical characteristics associated with the need for concomitant steroid eye drops administration were determined using logistic regression analysis. All patients were classified into 3 subgroups using cluster analysis. Results: PLC scores recorded in the sixth month were significantly improved compared with those recorded at baseline. PLC scores recorded in the 18th, 21st, and 24th months were significantly improved compared with those recorded in the sixth month. The remission rates increased diachronically and significantly, reaching 92% in the 24th month. Logistic regression analysis showed that, for every 10-year increase in patient age, the risk for requiring concomitant administration of steroid eye drops was reduced by half (odds ratio, 0.53; 95% confidence interval, 0.29–0.96). Using cluster analysis, the patients were divided into 3 clusters: adolescent type, pediatric type, and adult type. Conclusions: Two years of treatment with topical tacrolimus ophthalmic suspension is an effective method for inducing and maintaining the stable stages of VKC and AKC.

To investigate whether crude house-dust-mite antigen exacerbates eosinophilic inflammation in the conjunctival tissues of an atopic keratoconjunctivitis mouse model in a dose-dependent manner.An atopic keratoconjunctivitis mouse model was established by percutaneous sensitization and crude house-dust-mite antigen application in NC/Nga mice. To assess the dose-dependent response, conjunctival specimens from groups that were administered high- (High-HDM) or low-dose house-dust-mite antigen (Low-HDM) following percutaneous sensitization and the control without house-dust-mite antigen administration (control group) were evaluated. Histological examination and immunofluorescence staining were performed to determine eosinophil density and the number of IL-13-positive cells. Polymerase chain reaction array was used to obtain adaptive and innate immunity-related factor profile, and quantitative polymerase chain reaction was used to determine Il13, Il17a, Ccl11, and Ccl24 expression. Atopic keratoconjunctivitis model mice injected with anti-IL-1α antibody (IL-1α group) or vehicle (vehicle group) to the upper and lower eyelids before atopic keratoconjunctivitis development were evaluated.Eosinophil density in the conjunctiva increased with house-dust-mite antigen application in a dose-dependent manner. CD4, CXCL10, CCR6, C3, and IL-13 mRNA levels increased more than 5-fold in the conjunctiva of the High-HDM group animals compared to those in control animals. mRNA expression of Il13 and Ccl11 in the conjunctiva of the High-HDM group animals significantly increased compared with that in the Low-HDM and control group animals. Conversely, the eosinophil density and Il13 mRNA expression significantly decreased in the IL-1α group compared with those in the vehicle group.The house-dust-mite antigen increased eosinophilic infiltration and Il13 mRNA expression in the conjunctiva of an atopic keratoconjunctivitis mouse model in a dose-dependent manner. These inflammatory alterations were partially alleviated by eyelid injection of anti-IL-1α antibody. These findings indicate that IL-1α-induced IL-13 production constitutes a major exacerbating factor for house-dust-mite antigen-induced atopic keratoconjunctivitis.

We investigated ocular surface microbiota dysbiosis in patients with refractory allergic conjunctival diseases (ACDs; stratified into mild and severe groups) treated with topical tacrolimus.Patients (n = 21) with refractory ACDs (including vernal and atopic keratoconjunctivitis) actively treated with topical tacrolimus and 6 healthy controls were evaluated. Based on clinical scores and expression of specific cytokines on the ocular surface, patients with ACDs were divided into mild and severe groups using cluster analysis. The microbial composition of tear specimens collected from patients with mild and severe ACD and control subjects using the Schirmer test paper was determined through next-generation 16S rRNA sequencing analysis.Compared with healthy controls, patients with ACDs exhibited significantly decreased ocular surface microbiota α-diversity. Ocular surface microbiota mainly comprised members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria in all groups. The relative abundance of ocular surface microbiota in patients with ACDs was increased for phylum Firmicutes and decreased for phylum Proteobacteria (compared with control subjects). The genera Blautia (vs. mild ACD group) and Morganella (vs. control group) exhibited significantly increased abundance only in the severe ACD group.The ocular surface microbiota in patients with severe ACD exhibited decreased diversity and exacerbation of dysbiosis compared with that in patients with mild ACD and control subjects. Patients with mild refractory ACD also exhibited decreased diversity of these microbiota. These alterations in microbiota indicated a change in the ocular surface of patients with refractory ACD (be it because of disease pathogenesis or topical immunomodulatory treatment).

Endoreplication is a type of cell cycle where genome replication occurs without mitosis. An increase of ploidy level by endoreplication is often associated with cell enlargement and an enhanced plant growth. Here we report Arabidopsis thaliana subclass I ACTIN DEPOLYMERIZING FACTORs (ADFs) and vegetative ACTIN2/8 as novel regulators of endoreplication. A. thaliana has 11 ADF members that are divided into 4 subclasses. Subclass I consists of four members, ADF1, -2, -3, and -4, all of which constitutively express in various tissues. We found that both adf4 knockout mutant and transgenic plants in which expressions of all of four subclass I ADFs are suppressed (ADF1-4Ri) showed an increased leaf area of mature first leaves, which was associated with a significant increase of epidermal pavement cell area. Ploidy analysis revealed that the ploidy level was significantly increased in mature leaves of ADF1-4Ri. The increased ploidy was also observed in roots of adf4 and ADF1-4Ri, as well as in dark-grown hypocotyls of adf4. Furthermore, double mutants of vegetative ACT2 and ACT8 (act2/8) exhibited an increase of leaf area and ploidy level in mature leaves. Therefore, actin-relating pathway could regulate endoreplication. The possible mechanisms that actin and ADFs regulate endoreplication are discussed.

Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.

This study is a retrospectively recruited case series. We report three infants with acute conjunctivitis induced by β-lactamase–positive, ampicillin/clavulanic acid-resistant strains of Haemophilus influenzae (BLPACR). Patients with BLPACR-positive cultures were recruited from among 5,107 patients with inflammatory diseases of the ocular surface who underwent examinations, including bacterial culturing of conjunctival sac or corneal scrapings, between 2000 and 2015. Three BLPACR-positive patients were recruited, including a 10-month-old boy, a 4-month-old girl, and a 7-month-old girl. All three demonstrated BLPACR conjunctivitis. The clinical findings in these patients included fever, mucopurulent discharge, lid swelling, and conjunctival hyperemia. Samples of conjunctival swabs were obtained from all three infants, and BLPACR was isolated from all these conjunctival swabs. Antimicrobial susceptibility testing showed sensitivity to levofloxacin and resistance to ampicillin, cefaclor, and clarithromycin. We conclude that in infantile BLPACR conjunctivitis, simultaneous investigation for the determination of causative organism and antibiotic susceptibility testing are crucial aspects of the medical treatment.

Temperature is one of the most important of the physiological parameters that determine the biological status of living organisms. However, intracellular temperature was not imaged at the single-cell level until recently because of the lack of a molecular thermometer that can be applied to living cells. We have recently developed a method for imaging intracellular temperature using a cationic linear fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging microscopy (FLIM). The cationic linear FPT exhibits cell permeability in various mammalian cell lines and yeast cells, entering live cells within 10 min of incubation. Intracellular thermometry using the cationic linear FPT and FLIM can be used to image temperature with high temperature resolution (0.3-1.29 °C within a temperature range of 25-35 °C). The diffuse intracellular localization of the cationic linear FPT allows a high spatial resolution (i.e., the light microscope's diffraction limit, 200 nm), enabling the detection of temperature distributions at the subcellular level. This protocol, including the construction of a calibration curve and intracellular temperature imaging, requires ~14 h. Experience in handling cultured mammalian cells and use of a confocal laser-scanning microscope (CLSM) is required.

Purpose: We validated the use of chemokine messenger RNA (mRNA) expression analysis for the assessment of ocular surface allergic inflammation in chronic allergic conjunctival diseases (ACDs) with proliferative lesions, including giant papillae and gelatinous infiltration of the limbus. Methods: This prospective sectional study included 19 patients with chronic ACDs and 10 healthy volunteers as controls. Patients with chronic ACDs were divided into 2 subgroups according to the severity of the clinical score: active stage ACD subgroup (n = 9) and stable stage ACD subgroup (n = 10). Impression cytology using a filter paper for each upper tarsal conjunctiva of the patients with chronic ACDs and control subjects was performed, and the expression levels of IL1A, CXCL8, IL16, and CCL24 mRNAs encoding interleukin (IL)-1α, CXCL8/IL-8, IL-16, and CCL24/eotaxin-2, respectively, were determined by quantitative real-time polymerase chain reaction using impression cytology specimens. Results: CCL24 and IL16 mRNA levels in the active ACD subgroup were significantly higher than those in the control group (P = 0.003 and 0.004, respectively). IL1A and CXCL8 expression levels in the active ACD subgroup were significantly higher than those in the stable ACD (P = 0.008 and 0.029, respectively) and control (P = 0.008 and 0.014, respectively) subgroups. Furthermore, significant correlations were detected between IL16 and CCL24 mRNA levels (r = 0.76, P = 0.0001) and between IL1A and CXCL8 (r = 0.67, P = 0.0004). Conclusions: At least 2 kinds of inflammatory reactions, IL-1α- and CXCL8-associated inflammation and CCL24- and IL-16-associated inflammation, may be involved in the exacerbation of chronic ACDs.

Glycosylinositolphosphorylceramides (GIPCs) are the predominant lipid in the outer leaflet of the plasma membrane. Characterized GIPC glycosylation mutants have severe or lethal plant phenotypes. However, the function of the glycosylation is unclear. Previously, we characterized Arabidopsis thaliana GONST1 and showed that it was a nucleotide sugar transporter which provides GDP‐mannose for GIPC glycosylation. gonst1 has a severe growth phenotype, as well as a constitutive defense response. Here, we characterize a mutant in GONST1’s closest homolog, GONST2. The gonst2‐1 allele has a minor change to GIPC headgroup glycosylation. Like other reported GIPC glycosylation mutants, gonst1‐1gonst2‐1 has reduced cellulose, a cell wall polymer that is synthesized at the plasma membrane. The gonst2‐1 allele has increased resistance to a biotrophic pathogen Golovinomyces orontii but not the necrotrophic pathogen Botrytis cinerea. Expression of GONST2 under the GONST1 promoter can rescue the gonst1 phenotype, indicating that GONST2 has a similar function to GONST1 in providing GDP‐D‐Man for GIPC mannosylation.

Objective Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. Methods In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. Results In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by β3AR stimulation via a novel mechanism that involves Gβγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of β3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. Conclusions These data demonstrate that Gβγ-dependent Panx1 channel activation is involved in β3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of β3AR activation may have therapeutic implications., Highlights • Panx1 channel activation plays a critical role in thermogenic regulation in brown adipocytes. • Panx1 channel activity in brown adipocytes was induced by β3 adrenergic receptor stimulation. • Inactivation of Panx1 channels in cultured brown adipocytes resulted in defective cellular thermogenesis. • In mice, genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis. • Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis and aggravated diet-induced obesity and insulin resistance in mice.

Purpose. To evaluate the presence of ocular surface mucin in patients with atopic and vernal keratoconjunctivitis (AKC/VKC), we investigated the mRNA expression levels of SAM-pointed domain-containing ETS-like factor (SPDEF) and mucin-related genes on the ocular surface. Methods. Nineteen patients with AKC or VKC were divided into two groups based on the severity of the disease as determined by their clinical scores for AKC/VKC: the stable group and the active group. Impression cytology was performed in all patients using filter paper, and the expression levels of SPDEF, MUC1, MUC4, MUC5AC, MUC16, and eotaxin-2 mRNA were determined by real-time reverse-transcription polymerase chain reaction. Results. The results showed that the expression levels of SPDEF and MUC5AC mRNA in the active group were significantly decreased compared with those in the stable group. Furthermore, clinical scores were significantly negatively correlated with the expression levels of SPDEF mRNA and significantly positively correlated with the expression levels of eotaxin-2, which is a biomarker for eosinophilic inflammation on the ocular surface. Cluster analysis classified the patients with AKC/VKC into three clusters, and the stable group was divided into two clusters according to the condition of ocular surface mucin. Conclusions. Ocular surface mucin in patients with AKC/VKC is altered in accordance with the clinical severity of the disease.

PURPOSE To report an atypical presentation of herpes simplex virus (HSV) keratitis followed up using expression levels of HSV DNA in tears. METHODS A 22-year-old Japanese woman with hyperemia and foreign body sensation in her left eye was diagnosed with atypical dendritic keratitis. A slit-lamp examination at presentation indicated the presence of a rush of dendritic lesions with a sparse branching pattern and poor development of terminal bulbs; follicular conjunctivitis was also observed. Positivity for house-dust-mite- and cedar pollen-specific IgE antibodies in her serum indicated atopic diathesis. The HSV DNA levels in her tears were measured by a real-time polymerase chain reaction. RESULTS At the initial visit, the HSV DNA levels in tears were 6.4 × 10 copies/sample in the right eye and 1.6 × 10 copies/sample in the left eye. The keratitis improved after treatment with topical acyclovir ointment, 5 times a day for 7 days, and systemic valacyclovir 1000 mg/d for 5 days. Multiple punctate subepithelial opacities developed in her left eye on day 7, with undetectable HSV DNA in tears, bilaterally. CONCLUSIONS We have successfully monitored the HSV DNA levels in tears using quantitative real-time polymerase chain reaction in HSV keratitis where the corneal findings progressed from atypical dendritic keratitis to multiple punctate corneal subepithelial opacities during the treatment period.

Glycosylinositolphosphorylceramides (GIPCs) are the predominant lipid in the outer leaflet of the plasma membrane. Characterized GIPC glycosylation mutants have severe or lethal plant phenotypes. However, the function of the glycosylation is unclear. Previously, we characterized Arabidopsis thaliana GONST1 and showed that it was a nucleotide sugar transporter which provides GDP‐mannose for GIPC glycosylation. gonst1 has a severe growth phenotype, as well as a constitutive defense response. Here, we characterize a mutant in GONST1’s closest homolog, GONST2. The gonst2‐1 allele has a minor change to GIPC headgroup glycosylation. Like other reported GIPC glycosylation mutants, gonst1‐1gonst2‐1 has reduced cellulose, a cell wall polymer that is synthesized at the plasma membrane. The gonst2‐1 allele has increased resistance to a biotrophic pathogen Golovinomyces orontii but not the necrotrophic pathogen Botrytis cinerea. Expression of GONST2 under the GONST1 promoter can rescue the gonst1 phenotype, indicating that GONST2 has a similar function to GONST1 in providing GDP‐D‐Man for GIPC mannosylation.

A cationic fluorescent nanogel thermometer based on thermo-responsive N-isopropylacrylamide and environment-sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02-0.84 °C in the range 20-40 °C), and remarkable non-cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.

Background This study investigated the histamine H 1 and H 4 receptors mRNA ( H1R and H4R , respectively) expression on the ocular surface of patients with chronic forms of allergic conjunctival diseases to determine whether they can serve as biomarkers for allergic inflammation in the conjunctiva. Methods We examined 19 patients with vernal or atopic keratoconjunctivitis (AKC/VKC group) and 15 healthy volunteers (control group). The AKC/VKC group was divided into active and stable stage subgroups. Specimens were obtained from the upper tarsal conjunctiva of each participant using a modified impression cytology method. H1R , H4R , and eotaxin-1, -2, and -3 mRNA ( eotaxin-1 , eotaxin-2 , eotaxin-3 , respectively) expression was determined by real-time RT-PCR. Immunohistochemical analysis for eosinophil cationic protein (ECP), eosinophil major basic protein (MBP), eotaxin-2, and histamine H 4 receptor (H 4 R) were performed using conjunctival smears. Results The number of H4R -positive patients was higher in the active than the stable stage subgroup and control group, whereas no difference was observed for H1R . H1R levels were higher in the active than in the stable stage subgroup, while those of H4R were higher in the active stage subgroup than in the control group. H1R and H4R levels were correlated with eotaxin-2 level. In immunohistochemical analysis, H 4 R revealed their expression on eosinophils in conjunctival smears of patients with AKC/VKC. Conclusions H4R is useful as biomarkers of allergic inflammation on ocular surfaces. Most notably, H4R expressed on eosinophils is useful as a biomarker of eosinophilic inflammation of the ocular surface.

Glyceraldehyde-derived advanced glycation end products (glycer-AGE; also called Toxic-AGE [TAGE]) play a crucial role in the pathogenesis of diabetic angiopathy. However, the relationships between vitreous glycer-AGE levels and diabetic retinopathy (DR) severity, and between glycer-AGE levels and the levels of other angiogenic factors remain unknown. We investigated the correlation between levels of vitreous biomarkers, including glycer-AGE and angiogenic factors (vascular endothelial growth factor [VEGF], interleukin [IL]-8, leptin, placental growth factor [PlGF], endoglin, and fibroblast growth factor [FGF]-2) in patients with DR, using three DR staging groups. In this cross-sectional study, we examined 33 eyes from 33 patients with diabetes mellitus who underwent a vitrectomy (non-proliferative DR [NPDR, n = 8]; PDR with simple vitreous haemorrhage [VH, n = 17]; or PDR with a fibrovascular proliferative membrane [FVM, n = 8]). Vitreous levels of glycer-AGE and VEGF were evaluated using enzyme-linked immunosorbent assays. Vitreous levels of IL-8, leptin, PlGF, endoglin, and FGF-2 were evaluated using beaded assay methods. Vitreous levels of glycer-AGE in the FVM group were significantly higher than those in the NPDR and VH groups (all p

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein among eukaryotes. The main function of ADF is the severing and depolymerizing filamentous actin (F-actin), thus regulating F-actin organization and dynamics and contributing to growth and development of the organisms. Mammalian genomes contain only a few ADF genes, whereas angiosperm plants have acquired an expanding number of ADFs, resulting in the differentiation of physiological functions. Recent studies have revealed functions of ADFs in plant growth and development, and various abiotic and biotic stress responses. In biotic stress responses, ADFs are involved in both susceptibility and resistance, depending on the pathogens. Furthermore, recent studies have highlighted a new role of ADF in the nucleus, possibly in the regulation of gene expression. In this review, I will summarize the current status of plant ADF research and discuss future research directions.

RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

We performed simultaneous measurement of herpes simplex virus (HSV) DNA by real-time polymerase chain reaction (real-time PCR) and of HSV-specific secretory IgA antibody (HSV-sIgA) by enzyme-linked immunosorbent assay (ELISA) in tears obtained using Schirmer strips in order to investigate its diagnostic efficacy for herpes simplex keratitis (HSK). A total of 59 affected eyes from 59 patients with clinically suspected HSK (HSK group) and 23 eyes from 23 healthy volunteers (control group) were enrolled in this study. The HSK group was divided into five subgroups: dendritic/geographic keratitis, disciform keratitis, necrotizing keratitis, atypical keratitis, and others. The tear samples were taken using Schirmer strips to determine the HSV DNA and HSV-sIgA levels. The overall sensitivity and specificity were 55.8 and 100 % for HSV DNA and 49.2 and 82.6 % for HSV-sIgA. The HSV DNA levels in the disciform keratitis subgroup (median, 3.1 × 102 copies/sample) were significantly lower than those in the dendritic/geographic keratitis subgroup (median, 2.3 × 104 copies/sample) (P

Purpose: We report 2 patients with dry eye disease followed up using the expression levels of ocular surface mucin. Case Reports: Patient 1: a 57-year-old woman with Sjögren's syndrome-associated dry eyes experienced severe dryness and foreign body sensation in both her eyes, and instilled sodium hyaluronate ophthalmic solution 0.3% about 10-15 times daily. We measured the expression levels of MUC5AC mRNA (MUC5AC) and MUC16 mRNA (MUC16) by using real-time reversed transcription polymerase chain reaction for the specimens of modified impression cytology. Expression levels of MUC5AC and MUC16 on her ocular surface were very low. Subjective symptoms and expression levels of ocular surface mucin improved after combined treatment of rebamipide (4 times daily) and fluorometholone (once daily) ophthalmic suspension. Patient 2: a 62-year-old man with chronic graft-versus-host disease-associated dry eye experienced severe foreign body sensation and developed superficial punctate keratopathy with mucous thread and filamentary keratitis. Expression level of MUC5AC was very high at baseline. Subjective symptoms and expression levels of ocular surface mucin improved by combined treatment of rebamipide (4 times daily) and fluorometholone (once daily) ophthalmic suspension. Conclusion: Clinical test for MUC gene expression on the ocular surface was found to be useful in the follow-up of dry eye treatment.

Actin-depolymerizing factors (ADFs) are conserved proteins that function in regulating the structure and dynamics of actin microfilaments in eukaryotes. In this study, we present evidence that Arabidopsis (Arabidopsis thaliana) subclass I ADFs, particularly ADF4, functions as a susceptibility factor for an adapted powdery mildew fungus. The null mutant of ADF4 significantly increased resistance against the adapted powdery mildew fungus Golovinomyces orontii. The degree of resistance was further enhanced in transgenic plants in which the expression of all subclass I ADFs (i.e. ADF1–ADF4) was suppressed. Microscopic observations revealed that the enhanced resistance of adf4 and ADF1-4 knockdown plants (ADF1-4Ri) was associated with the accumulation of hydrogen peroxide and cell death specific to G. orontii-infected cells. The increased resistance and accumulation of hydrogen peroxide in ADF1-4Ri were suppressed by the introduction of mutations in the salicylic acid- and jasmonic acid-signaling pathways but not by a mutation in the ethylene-signaling pathway. Quantification by microscopic images detected an increase in the level of actin microfilament bundling in ADF1-4Ri but not in adf4 at early G. orontii infection time points. Interestingly, complementation analysis revealed that nuclear localization of ADF4 was crucial for susceptibility to G. orontii. Based on its G. orontii-infected-cell-specific phenotype, we suggest that subclass I ADFs are susceptibility factors that function in a direct interaction between the host plant and the powdery mildew fungus.

Obligate biotrophic fungi that cause powdery mildew on host plants form a specialized infection organ called the haustorium in the host apoplast. It was previously reported that the haustorium is surrounded by host actin microfilaments (AFs). The previous study used fixed cells, in which AFs were stained with fluorescently labeled phalloidine, therefore the structural dynamics of haustorium-surrounding AFs has not been examined. In the present study, we performed a live imaging analysis to examine the dynamics and developmental changes in the organization of haustorium-surrounding host AFs using host Arabidopsis thaliana and A. thaliana-adapted powdery mildew fungus Golovinomyces orontii. Image correlation-based velocimetry analysis suggested that AFs around haustorium are rather static compared to the dynamicity of AFs at the cell surface. Quantification of AF density and bundling showed that the density, but not the level of bundling, of haustorium-surrounding AFs increased as the haustorium matures. The possible role of AFs around haustoria is discussed.

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.

The Golgi lumen is the site of many different glycosylation events, including cell wall polysaccharide biosynthesis and lipid glycosylation. Transporters are necessary for the import of the substrates required for glycosylation (nucleotide sugars) from the cytosol where they are synthesized. Plants use four GDP-linked sugars to glycosylate macromolecules: GDP-L-Fucose, GDP-D-Mannose, GDP-L-Galactose and GDP-D-Glucose. Of the predicted fifty-one members of the nucleotide sugar transporter/triose phosphate transporter family in Arabidopsis, only four appear to contain the conserved motif needed for the transport of GDP-linked sugars, GOLGI LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST) 1-4. Previously, we have demonstrated that GONST1 provides GDP-D-Mannose for glycosylation of a class of sphingolipids, the glycosylinositolphosphorylceramides (GIPCs). Here, we characterize its closest homologue, GONST2, and conclude that it also specifically provides substrate for GIPC glycosylation. Expression ofGONST2driven by theGONST1promoter is able to rescue the severe growth phenotype ofgonst1. Loss of GONST2 exacerbates thegonst1constitutive hypersensitive response, as well as the reduced cell wall cellulose content. Thegonst2mutant grows normally under standard conditions, but has enhanced resistance to the powdery mildew-causing fungusGolovinomyces orontii.

Digitization of clinical observation is necessary for assessing the severity of superior limbic keratoconjunctivitis (SLK). This study aimed to use a novel quantitative marker to examine hyperemia in patients with SLK.We included six eyes of six patients with both dry eye disease and SLK (SLK group) and eight eyes of eight patients with Sjögren syndrome (SS group). We simultaneously obtained the objective finding scores by using slit-lamp examination and calculated the superior hyperemia index (SHI) with an automated conjunctival hyperemia analysis software by using photographs of the anterior segment. Three objective finding scores, including papillary formation of the superior palpebral conjunctiva, superior limbal hyperemia and swelling, and superior corneal epitheliopathy, were determined. The SHI was calculated as the superior/temporal ratio of bulbar conjunctival hyperemia by using the software. Fisher's exact test was used to compare a high SHI (≥1.07) ratio between the SLK and SS groups. P-Values0.05 were considered statistically significant.The SHI (mean ± standard deviation) in the SLK and SS groups was 1.19 ± 0.50 and 0.69 ± 0.24, respectively. The number of patients with a high SHI (≥1.07) was significantly higher in the SLK group than in the SS group (p 0.05). The sensitivity and specificity of the SHI in the differential diagnosis between SS and SLK were 66.7% and 87.5%, respectively. An analysis of the association between the objective finding scores and SHI showed that the SHI had a tendency to indicate the severity of superior limbal hyperemia and swelling score in the SLK group.The SHI calculated using the automated conjunctival hyperemia analysis software could successfully quantify superior bulbar conjunctival hyperemia and may be a useful tool for the differential diagnosis between SS and SLK and for the quantitative follow-up of patients with SLK.

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.

To retrospectively investigate the clinical characteristics of Moraxella keratitis.We reviewed the medical records of 17 patients with Moraxella keratitis. Onset age, sex, predisposing factors, initial clinical presentations, culture and antimicrobial susceptibility testing, and management and outcome of medical treatment were investigated.Moraxella keratitis was more common in patients older than 40 years of age, and its representative initial presentation was a round-shaped ulcer with endothelial plaque (70.6%) and hypopyon (58.8%). Local predisposing factors were significantly more frequent than systemic predisposing factors (P 0.005). Isolated strains of Moraxella (M. catarrhalis, M. osloensis, and other Moraxella spp.) were sensitive to all antibiotics tested except ampicillin. The common disease contraction period was2 weeks.Moraxella keratitis (including the first report of M. osloensis keratitis) had local predisposing factors, high sensitivity to antibiotics, and a tendency to recover within 2 weeks.