Interaction of EXA1 and eIF4E Family Members Facilitates Potexvirus Infection in Arabidopsis thaliana.

Plant viruses depend on a number of host factors for successful infection. Deficiency of critical host factors confers recessively inherited viral resistance in plants. For example, loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. However, the molecular mechanism of how EXA1 assists potexvirus infection remains largely unknown. Previous studies reported that the salicylic acid (SA) pathway is upregulated in exa1 mutants, and EXA1 modulates hypersensitive response-related cell death during EDS1-dependent effector-triggered immunity. Here, we show that exa1-mediated viral resistance is mostly independent of SA and EDS1 pathways. We demonstrate that Arabidopsis EXA1 interacts with three members of the eukaryotic translation initiation factor 4E (eIF4E) family, eIF4E1, eIFiso4E, and novel cap-binding protein (nCBP), through the eIF4E-binding motif (4EBM). Expression of EXA1 in exa1 mutants restored infection by the potexvirus Plantago asiatica mosaic virus (PlAMV), but EXA1 with mutations in 4EBM only partially restored infection. In virus inoculation experiments using Arabidopsis knockout mutants, EXA1 promoted PlAMV infection in concert with nCBP, but the functions of eIFiso4E and nCBP in promoting PlAMV infection were redundant. By contrast, the promotion of PlAMV infection by eIF4E1 was, at least partially, EXA1 independent. Taken together, our results imply that the interaction of EXA1-eIF4E family members is essential for efficient PlAMV multiplication, although specific roles of three eIF4E family members in PlAMV infection differ. IMPORTANCE The genus Potexvirus comprises a group of plant RNA viruses, including viruses that cause serious damage to agricultural crops. We previously showed that loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. EXA1 may thus play a critical role in the success of potexvirus infection; hence, elucidation of its mechanism of action is crucial for understanding the infection process of potexviruses and for effective viral control. Previous studies reported that loss of EXA1 enhances plant immune responses, but our results indicate that this is not the primary mechanism of exa1-mediated viral resistance. Here, we show that Arabidopsis EXA1 assists infection by the potexvirus Plantago asiatica mosaic virus (PlAMV) by interacting with the eukaryotic translation initiation factor 4E family. Our results imply that EXA1 contributes to PlAMV multiplication by regulating translation.

Diaphragm Ultrasonography: Reference Values and Influencing Factors for Thickness, Thickening Fraction, and Excursion in the Seated Position.

INTRODUCTION: Measurements of diaphragm function by ultrasonography are affected by body position, but reference values in the seated position have not been established for an Asian population. This study aimed to determine reference values for diaphragm thickness, thickening fraction, and dome excursion by ultrasonography and to investigate the effects of sex, height, and body mass index. METHODS: Diaphragm ultrasonography was performed on 109 seated Japanese volunteers with normal respiratory function who were enrolled between March 2022 and January 2023. Thickness, thickening fraction, and excursion were measured. Reference values and the measurement success rate were calculated. Multivariate analysis adjusted for sex, height, and body mass index was performed. RESULTS: The measurement success rate was better for thickness than for excursion. The mean (lower limit of normal) values on the right/left sides were as follows. During quiet breathing, thickness at end expiration(mm) was 1.7 (0.9)/1.6 (0.80), thickening fraction(%) was 50 (0.0)/52 (0.0), and excursion(cm) was 1.7 (0.5)/1.9 (0.5). During deep breathing, the thickening fraction was 111 (24)/107 (22), and the excursion was 4.4 (1.7)/4.1 (2.0). In multivariate analysis, body mass index was positively associated with thickness but not with the thickening fraction. CONCLUSION: The reference values in this study were smaller than those in previous reports from Europe. Considering that thickness is influenced by body mass index, using Western reference values in Asia, where the average body mass index is lower, might not be appropriate. The thickening fraction in deep breathing is unaffected by other items and can be used more universally.

Relationship Between Diaphragm Thickness, Thickening Fraction, Dome Excursion, and Respiratory Pressures in Healthy Subjects: An Ultrasound Study.

PURPOSE: Diaphragm ultrasonography is used to identify causes of diaphragm dysfunction. However, its correlation with pulmonary function tests, including maximal inspiratory (MIP) and expiratory pressures (MEP), remains unclear. This study investigated this relationship by measuring diaphragm thickness, thickening fraction (TF), and excursion (DE) using ultrasonography, and their relationship to MIP and MEP. It also examined the influence of age, sex, height, and BMI on these measures. METHODS: We recruited healthy Japanese volunteers and conducted pulmonary function tests and diaphragm ultrasonography in a seated position. Diaphragm ultrasonography was performed during quiet breathing (QB) and deep breathing (DB) to measure the diaphragm thickness, TF, and DE. A multivariate analysis was conducted, adjusting for age, sex, height, and BMI. RESULTS: Between March 2022 and January 2023, 109 individuals (56 males) were included from three facilities. The mean (standard deviation) MIP and MEP [cmH2O] were 72.2 (24.6) and 96.9 (35.8), respectively. Thickness [mm] at the end of expiration was 1.7 (0.4), TF [%] was 50.0 (25.9) during QB and 110.7 (44.3) during DB, and DE [cm] was 1.7 (0.6) during QB and 4.4 (1.4) during DB. Multivariate analysis revealed that only DE (DB) had a statistically significant relationship with MIP and MEP (p = 0.021, p = 0.008). Sex, age, and BMI had a statistically significant influence on relationships between DE (DB) and MIP (p = 0.008, 0.048, and < 0.001, respectively). CONCLUSION: In healthy adults, DE (DB) has a relationship with MIP and MEP. Sex, age, and BMI, but not height, are influencing factors on this relationship.

Short 5' Untranslated Region Enables Optimal Translation of Plant Virus Tricistronic RNA via Leaky Scanning.

Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by readthrough of upstream ORFs, has great potential to translate polycistronic mRNAs. However, the mRNA elements controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate thatthe significantly shorter 5' untranslated region (UTR) of the most upstream ORF promotes leaky scanning, potentially fine-tuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Our results suggest that the remarkably short length of the leader sequence, like the Kozak sequence, is a translational regulatory element with a biologically important role, as previous studies have shown biochemically. IMPORTANCEPotexvirus, a group of plant viruses, infect a variety of crops, including cultivated crops. It has been thought that the three transition proteins that are essential for the cell-to-cell transfer of potexviruses are translated from two subgenomic RNAs, sgRNA1 and sgRNA2. However, sgRNA2 has not been clearly detected. In this study, we have shown that sgRNA1, but not sgRNA2, is the major translation template for the three movement proteins. In addition, we determined the transcription start site of sgRNA1 in flexiviruses and found that the efficiency of leaky scanning caused by the short 5' UTR of sgRNA1, a widely conserved feature, regulates the translation of the three movement proteins. When we tested the infection of viruses with mutations introduced into the length of the 5' UTR, we found that the movement efficiency of the virus was affected. Our results provide important additional information on the protein translation strategy of flexiviruses, including Potexvirus, and provide a basis for research on their control as well as the need to reevaluate the short 5' UTR as a translational regulatory element with an important role in vivo.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota.

Powdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored. We performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs). We found that after infection of soil-grown plants, G. orontii outcompeted numerous resident leaf-associated fungi while fungal community structure in roots remained unaltered. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-derived SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root-derived SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a twofold increase in leaf bacterial load. Our findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status.

[Recessive resistance to plant viruses by the deficiency of eukaryotic translation initiation factor genes.]

Plant viruses, obligate parasitic pathogens, utilize a variety of host plant factors in the process of their infection due to the limited number of genes encoded in their own genomes. The genes encoding these host factors are called susceptibility genes because they are responsible for the susceptibility of plants to viruses. Plants lacking or having mutations in a susceptibility gene essential for the infection of a virus acquire resistance to the virus. Such resistance trait is called recessive resistance because of the recessive inherited characteristics. Recessive resistance is reported to account for about half of the plant viral resistance loci mapped in known cultivated crops. Eukaryotic translation initiation factor (eIF) 4E family genes are well-known susceptibility genes. Although there are many reports about eIF4E-mediated recessive resistance to plant viruses, the mechanistic insight of the resistance is still limited. Here we review focusing on studies that have elucidated the mechanism of eIF4E-mediated recessive resistance.

Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization.

Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.

Comprehensive screening of antimicrobials to control phytoplasma diseases using an in vitro plant-phytoplasma co-culture system.

Phytoplasmas are plant-pathogenic bacteria that infect many important crops and cause serious economic losses worldwide. However, owing to an inability to culture phytoplasmas, screening of antimicrobials on media is difficult. The only antimicrobials being used to control phytoplasmas are tetracycline-class antibiotics. In this study, we developed an accurate and efficient screening method to evaluate the effects of antimicrobials using an in vitro plant-phytoplasma co-culture system. We tested 40 antimicrobials, in addition to tetracycline, and four of these (doxycycline, chloramphenicol, thiamphenicol and rifampicin) decreased the accumulation of 'Candidatus (Ca.) Phytoplasma asteris'. The phytoplasma was eliminated from infected plants by the application of both tetracycline and rifampicin. We also compared nucleotide sequences of rRNAs and amino acid sequences of proteins targeted by antimicrobials between phytoplasmas and other bacteria. Since antimicrobial target sequences were conserved among various phytoplasma species, the antimicrobials that decreased accumulation of 'Ca. P. asteris' may also have been effective against other phytoplasma species. These approaches will provide new strategies for phytoplasma disease management.

EXA1, a GYF domain protein, is responsible for loss-of-susceptibility to plantago asiatica mosaic virus in Arabidopsis thaliana.

One of the plant host resistance machineries to viruses is attributed to recessive alleles of genes encoding critical host factors for virus infection. This type of resistance, also referred to as recessive resistance, is useful for revealing plant-virus interactions and for breeding antivirus resistance in crop plants. Therefore, it is important to identify a novel host factor responsible for robust recessive resistance to plant viruses. Here, we identified a mutant from an ethylmethane sulfonate (EMS)-mutagenized Arabidopsis population which confers resistance to plantago asiatica mosaic virus (PlAMV, genus Potexvirus). Based on map-based cloning and single nucleotide polymorphism analysis, we identified a premature termination codon in a functionally unknown gene containing a GYF domain, which binds to proline-rich sequences in eukaryotes. Complementation analyses and robust resistance to PlAMV in a T-DNA mutant demonstrated that this gene, named Essential for poteXvirus Accumulation 1 (EXA1), is indispensable for PlAMV infection. EXA1 contains a GYF domain and a conserved motif for interaction with eukaryotic translation initiation factor 4E (eIF4E), and is highly conserved among monocot and dicot species. Analysis using qRT-PCR and immunoblotting revealed that EXA1 was expressed in all tissues, and was not transcriptionally responsive to PlAMV infection in Arabidopsis plants. Moreover, accumulation of PlAMV and a PlAMV-derived replicon was drastically diminished in the initially infected cells by the EXA1 deficiency. Accumulation of two other potexviruses also decreased in exa1-1 mutant plants. Our results provided a functional annotation to GYF domain-containing proteins by revealing the function of the highly conserved EXA1 gene in plant-virus interactions.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor.

RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

Nucleocapsid protein from fig mosaic virus forms cytoplasmic agglomerates that are hauled by endoplasmic reticulum streaming.

UNLABELLED: Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE: Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification.

Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

Lectin-mediated resistance impairs plant virus infection at the cellular level.

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.

Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus, a unique tobamovirus that contains an internal poly(A) region in its 3' end.

In this study, we detected a Japanese isolate of hibiscus latent Fort Pierce virus (HLFPV-J), a member of the genus Tobamovirus, in a hibiscus plant in Japan and determined the complete sequence and organization of its genome. HLFPV-J has four open reading frames (ORFs), each of which shares more than 98 % nucleotide sequence identity with those of other HLFPV isolates. Moreover, HLFPV-J contains a unique internal poly(A) region of variable length, ranging from 44 to 78 nucleotides, in its 3'-untranslated region (UTR), as is the case with hibiscus latent Singapore virus (HLSV), another hibiscus-infecting tobamovirus. The length of the HLFPV-J genome was 6431 nucleotides, including the shortest internal poly(A) region. The sequence identities of ORFs 1, 2, 3 and 4 of HLFPV-J to other tobamoviruses were 46.6-68.7, 49.9-70.8, 31.0-70.8 and 39.4-70.1 %, respectively, at the nucleotide level and 39.8-75.0, 43.6-77.8, 19.2-70.4 and 31.2-74.2 %, respectively, at the amino acid level. The 5'- and 3'-UTRs of HLFPV-J showed 24.3-58.6 and 13.0-79.8 % identity, respectively, to other tobamoviruses. In particular, when compared to other tobamoviruses, each ORF and UTR of HLFPV-J showed the highest sequence identity to those of HLSV. Phylogenetic analysis showed that HLFPV-J, other HLFPV isolates and HLSV constitute a malvaceous-plant-infecting tobamovirus cluster. These results indicate that the genomic structure of HLFPV-J has unique features similar to those of HLSV. To our knowledge, this is the first report of the complete genome sequence of HLFPV.

Relationship between the characteristics of Japanese physicians involved in medical care for older adults and their approaches to treating older patients with multimorbidity.

One countermeasure against the increasing prevalence of multimorbidity is the need to provide clinical education and training that considers the characteristics of physicians. We conducted a questionnaire survey to determine the relationship between physicians' characteristics and their approach to treating older patients with multimorbidity. A total of 3300 geriatric specialists and primary care specialists in Japan were enrolled. A 4-point Likert scale was used to score the following items: difficult diseases (43 items), difficult patient backgrounds (14 items), important clinical factors (32 items), and important clinical management (32 items). Exploratory factor analysis was performed to examine the constructs in each of the scales Diseases, Backgrounds, Clinical Factors, and Clinical Management, and group comparisons by physician characteristics were conducted. A total of 778 respondents were included in the analysis. Six factors for Diseases, two factors for Patient Background, four factors for Clinical Factors, and two factors for Clinical Management were explored as patterns. Group comparison between mean scores for each factor and the characteristics of responding physicians showed statistically significant differences in at least one factor for all patterns in terms of years of experience as a physician (26 years or less, 27 years or more), the clinical setting (providing or not providing home medical care), and sex (male or female). Our results suggest a need for clinical education and training that takes into account not only physicians' experience and clinical setting, but also their sex.

Exercise-induced vitamin D receptor and androgen receptor mediate inhibition of IL-6 and STAT3 in muscle.

Background: Skeletal muscle produces interleukin-6 (IL-6) during exercise as a myokine. Although IL-6 is required for skeletal muscle regeneration, its action increases the expression of myostatin and other proteins involved in muscle atrophy, resulting in skeletal muscle atrophy. In this study, we clarified the effects exercise-induced vitamin D receptor (VDR) and androgen receptor (AR) expression on IL-6 and signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Method: C2C12 myotubes were subjected to electric pulse stimulation (EPS) in vitro. To evaluate VDR and AR function, a VDR/AR agonist and antagonist were administered before EPS to C2C12 myotubes. C57BL6 mice underwent 4 weeks of exercise. The expression levels of proteolytic-associated genes, including CCAAT/enhancer-binding protein delta (C/EBPδ) and myostatin, were measured by quantitative real-time polymerase chain reaction, and phosphorylated and total STAT3 levels were measured by Western blot analysis. Result: The expression of VDR and AR mRNA was induced following EPS in C2C12 myotubes. IL-6 mRNA expression was also increased with a peak at 6 h after EPS and p-STAT3/STAT3 ratio reciprocally decreased. Although VDR/AR agonist administration decreased IL-6 mRNA expression and p-STAT3/STAT3 ratio, these two endpoints increased after treatment with VDR/AR antagonist, respectively. Exercise in mice also increased the expression of VDR/AR and IL-6 mRNA and decreased p-STAT3/STAT3 ratio. Conclusion: Exercise-induced VDR and AR expression results in the suppression of IL-6 mRNA and STAT3 phosphorylation in skeletal muscle.

Facilitators, barriers, and changes in POCUS use: longitudinal follow-up after participation in a national point-of-care ultrasound training course in Japan.

BACKGROUND: POCUS training courses are effective at improving knowledge and skills, but few studies have followed learners longitudinally post-course to understand facilitators, barriers, and changes in POCUS use in clinical practice. We conducted a prospective observational study of physicians who attended 11 standardized POCUS training courses between 2017 and 2019 in Japan. Physicians who attended a standardized POCUS course were surveyed about their current frequency of POCUS use of the heart, lung, abdomen, and lower extremity veins, and perceived barriers and facilitators to POCUS use in clinical practice. RESULTS: Data were analyzed from 112 completed surveys (response rate = 20%). A majority of responding physicians were faculty (77%) in internal medicine (69%) affiliated with community hospitals (55%). The mean delay between course attendance and survey response was 50.3 months. A significant increase in POCUS use from < 1 to ≥ 1 time per week was seen for all organ systems after 50 months post-course (p < 0.01). Approximately half of course participants reported an increase in the frequency of cardiac (61%), lung (53%), vascular (44%), and abdominal (50%) ultrasound use. General facilitators of POCUS use were easy access to ultrasound machines (63%), having a colleague with whom to learn POCUS (47%), and adequate departmental support (46%). General barriers included lack of opportunities for POCUS training (47%), poor access to ultrasound machines (38%), and limited time for POCUS training (33%). In the group with increased POCUS usage, specific facilitators reported were enhanced POCUS knowledge, improved image acquisition skills, and greater self-confidence in performing POCUS. Conversely, the group without increased POCUS usage reported lack of supervising physicians, low confidence, and insufficient training opportunities as specific barriers. CONCLUSIONS: Approximately half of physicians reported an increase in cardiac, lung, vascular, and abdominal POCUS use > 4 years after attending a POCUS training course. In addition to improving access to ultrasound machines and training opportunities, a supportive local clinical environment, including colleagues to share experiences in learning POCUS and local experts to supervise scanning, is important to foster ongoing POCUS practice and implementation into clinical practice.

Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement.

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.