A Putative Apoplastic Effector of Clavibacter capsici, ChpGCc as Hypersensitive Response and Virulence (Hrv) Protein in Plants.

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide-deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Analysis of particulate matter-induced alteration of genes and related signaling pathways in the respiratory system.

Airborne particulate matter (PM) is a global environmental risk factor threatening human health and is a major cause of cardiovascular and respiratory disease-associated death. Current studies on PM exposure have been limited to large-scale cohort and epidemiological investigations, emphasizing the need for detailed individual-level studies to uncover specific differentially expressed genes and their associated signaling mechanisms. Herein, we revealed that PM exposure significantly upregulated inflammatory and immune responses, such as cytokine-mediated signaling pathways, complement system, and the activation and migration of immune cells in gene set enrichment analysis of our RNA sequencing (RNAseq) data. Remarkably, we discovered that the broad gene expression and signaling pathways mediated by macrophages were predominantly expressed in the respiratory system following PM exposure. Consistent with these observations, individual PMs, classified by aerodynamic size and origin, significantly promoted macrophage recruitment to the lungs in the mouse lung inflammation model. Additionally, we confirmed that RNAseq observations from the respiratory system were reproduced in murine bone marrow-derived macrophages and the alveolar macrophage cell line MH-S after individual PM exposure. Our findings demonstrated that PM exposure augmented broad inflammatory and immune responses in the respiratory system and suggested the reinforcement of global strategies for reducing particulate air pollution to prevent respiratory diseases and their exacerbation.

The alternative bile acid pathway can predict food allergy persistence in early childhood.

BACKGROUND: Mechanisms underlying persistent food allergy (FA) are not well elucidated. The intestinal mucosa is the primary exposure route of food allergens. However, no study has examined intestinal metabolites associated with FA persistence. The goal of this study was to investigate intestinal metabolites and associated microbiomes in early life that aid in determining the development and persistence of FA. METHODS: We identified metabolomic alterations in the stool of infants according to FA by mass spectrometry-based untargeted metabolome profiling. The targeted metabolomic analysis of bile acid metabolites and stool microbiome was performed. Bile acid metabolite composition in infancy was evaluated by characterizing the subjects at the age of 3 into FA remission and persistent FA. RESULTS: In untargeted metabolomics, primary bile acid biosynthesis was significantly different between subjects with FA and healthy controls. In targeted metabolomics for bile acids, intestinal bile acid metabolites synthesized by the alternative pathway were reduced in infants with FA than those in healthy controls. Subjects with persistent FA were also distinguished from healthy controls and those with FA remission by bile acid metabolites of the alternative pathway. These metabolites were negatively correlated with specific IgE levels in egg white. The abundance of intestinal Clostridia was decreased in the FA group and was correlated with ursodeoxycholic acid. CONCLUSION: Intestinal bile acid metabolites of the alternative pathway could be predictive biomarkers for persistent FA in early childhood. These findings require replication in future studies.

First report of Soybean mosaic virus of sword bean in South Korea.

Soybean mosaic virus (SMV) is a member of the genus Potyvirus in the family Potyviridae. Legume crops are often infected by SMV. SMV has not been naturally isolated from sword bean (Canavalia gladiata) in South Korea. In July 2021, 30 samples of sword bean were collected at the field located in Hwasun and Muan, Jeonnam, Korea to investigate viruses infecting sword bean. The samples exhibited symptoms typical of viral infection such as mosaic pattern and, mottling of leaves. Reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) techniques were employed to identify the agent of viral infection in sword bean samples. Total RNA was extracted from the samples using the Easy-SpinTM Total RNA Extraction Kit (Intron, Seongnam, Korea). Out of the 30 samples, seven were found to be infected by the SMV. RT-PCR was performed using RT-PCR Premix (GeNet Bio, Daejeon, Korea) with SMV-specific primer set, forward primer (SM-N40, 5'-CATATCAGTTTGTTGGGCA-3') and the reverse primer (SM-C20, 5'-TGCCTATACCCTCAACAT-3'), yielding a product of 492 bp (Lim et al., 2014). RT-LAMP was performed using RT-LAMP Premix (EIKEN Chemical, Tokyo, Japan) with SMV-specific primer set, the forward primer (SML-F3, 5'-GACGATGAACAGATGGGC-3', SML-FIP, 5'-GCATCTGGAGATGTGCTTTTGTGGTTATGAATGGTTTCATGG-3') and reverse primer (SML-B3, 5'-TCTCAGAGTTGGTTTTGCA-3', SML-BIP, 5'-GCGTGTGGGTGATGATGGATTTTTTCGACAATGGGTTTCAGC-3') for diagnosis of viral infection (Lee et al., 2015). The full coat protein genes of seven isolates were amplified using RT-PCR to determine their nucleotide sequence. The standard nucleotide BLAST (blastn suite) showed that the seven isolates had approximately 98.2-100% homology with SMV isolates (FJ640966, MT603833, MW079200, and MK561002) in NCBI GenBank. The sequences of seven isolates were deposited in the GenBank database under the accession numbers: OP046403-9. For the pathogenicity assay of the isolate, the crude saps from SMV-infected samples were mechanically inoculated into sword bean. Fourteen days after inoculation, the mosaic symptoms were observed on the upper leaves of sword bean. As a result of the RT-PCR diagnosis in the upper leaves, SMV was reconfirmed in sword bean. This is the first report of natural SMV infection in sword bean. As sword beans are increasingly consumed for teas, transmitted seeds are resulting in a decrease in pod production and quality. It is necessary to develop efficient methods of seed processing and management strategies to control SMV infection in sword bean.

Exploring the Anti-Osteoporotic Potential of Daucosterol: Impact on Osteoclast and Osteoblast Activities.

Osteoporosis is a debilitating condition characterized by reduced bone mass and density, leading to compromised structural integrity of the bones. While conventional treatments, such as bisphosphonates and selective estrogen receptor modulators (SERMs), have been employed to mitigate bone loss, their effectiveness is often compromised by a spectrum of adverse side effects, ranging from gastrointestinal discomfort and musculoskeletal pain to more severe concerns like atypical fractures and hormonal imbalances. Daucosterol (DC), a natural compound derived from various plant sources, has recently garnered considerable attention in the field of pharmacology. In this study, we investigated the anti-osteoporosis potential of DC by characterizing its role in osteoclasts, osteoblasts, and lipopolysaccharide (LPS)-induced osteoporosis. The inhibitory effect of DC on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation by fluorescent staining, and bone resorption by pit formation assay. In addition, the calcification nodule deposition effect of osteoblasts was determined by Alizarin red S staining. The effective mechanisms of both cells were verified by Western blot and reverse transcription polymerase chain reaction (RT-PCR). To confirm the effect of DC in vivo, DC was administered to a model of osteoporosis by intraperitoneal administration of LPS. The anti-osteoporosis effect was then characterized by micro-CT and serum analysis. The results showed that DC effectively inhibited osteoclast differentiation at an early stage, promoted osteoblast activity, and inhibited LPS-induced bone density loss. The results of this study suggest that DC can treat osteoporosis through osteoclast and osteoblast regulation, and therefore may be considered as a new therapeutic alternative for osteoporosis patients in the future.

Exosome from IFN-γ-Primed Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Improved Skin Inflammation and Barrier Function.

The pathogenesis of atopic dermatitis (AD) is multifactorial, including immune dysregulation and epidermal barrier defects, and a novel therapeutic modality that can simultaneously target multiple pathways is needed. We investigated the therapeutic effects of exosomes (IFN-γ-iExo) secreted from IFN-γ-primed induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) in mice with Aspergillus fumigatus-induced AD. IFN-γ-iExo was epicutaneously administered to mice with AD-like skin lesions. The effects of IFN-γ-iExo treatment were investigated through clinical scores, transepidermal water loss (TEWL) measurements, and histopathology. To elucidate the therapeutic mechanism, we used an in vitro model of human keratinocyte HaCaT cells stimulated with IL-4 and IL-13 and performed extensive bioinformatics analysis of skin mRNA from mice. The expression of indoleamine 2,3-dioxygenase was higher in IFN-γ primed iMSCs than in iMSCs. In human keratinocyte HaCaT cells, treatment with IFN-γ-iExo led to decreases in the mRNA expression of thymic stromal lymphopoietin, IL-25, and IL-33 and increases in keratin 1, keratin 10, desmoglein 1, and ceramide synthase 3. IFN-γ-iExo treatment significantly improved clinical and histological outcomes in AD mice, including clinical scores, TEWL, inflammatory cell infiltration, and epidermal thickness. Bioinformatics analysis of skin mRNA from AD mice showed that IFN-γ-iExo treatment is predominantly involved in skin barrier function and T cell immune response. Treatment with IFN-γ-iExo improved the clinical and histological outcomes of AD mice, which were likely mediated by restoring proper skin barrier function and suppressing T cell-mediated immune response.

Combined effect of hygienic and polygenic risk scores in children with allergic rhinitis.

BACKGROUND: Although the development of allergic rhinitis (AR) is associated with multiple genetic and hygienic environmental factors, previous studies have focused mostly on the effect of a single factor on the development of AR. OBJECTIVE: This study aimed to investigate the combined effect of multiple genetic and hygienic environmental risk factors on AR development in school children. METHODS: We conducted a cross-sectional study, comprising 1,797 children aged 9-12 years. Weighted environmental risk score (ERS) was calculated by using four hygienic environmental factors, including antibiotic use during infancy, cesarean section delivery, breast milk feeding, and having older siblings. Weighted polygenic risk score (PRS) was calculated by using four single nucleotide polymorphisms (SNPs), including interleukin-13 (rs20541), cluster of differentiation 14 (rs2569190), toll-like receptor 4 (rs1927911), and glutathione S-transferase P1 (rs1695). Multivariable logistic regression analysis was used. RESULTS: More than three courses of antibiotic use during infancy increased the risk of current AR (adjusted odd ratio [aOR], 2.058; 95% confidence interval [CI]: 1.290-3.284). Having older siblings, especially > 2 (aOR, 0.526; 95%Cl: 0.303-0.913) had a protective effect. High ERS ( > median; aOR, 2.079; 95%Cl: 1.466-2.947) and PRS ( > median; aOR, 1.627; 95%Cl: 1.117-2.370) increased the risk of current AR independently. Furthermore, children who had both high ERS and PRS showed a higher risk of current AR (aOR, 3.176; 95%Cl: 1.787-5.645). CONCLUSIONS: Exposure to multiple hygienic risk factors during infancy increases the risk of AR in genetically susceptible children.

High-Resolution Surface Replication of Living Organisms using Air-Through-Precursor Suction-Augmented Replica Molding.

Replica molding is widely used to reproduce the surface microstructures that provide living organisms with distinct and useful functions. However, the existing methods are limited by the low resolution resulting from the air trapped in the structures during precursor solution loading. This study investigated replica molding with an air-through-precursor suction (APS) process, which used a degassed polydimethylsiloxane substrate to remove the trapped air through the precursor solution. The liquid loading times are characterized using a model template, and air suction that is up to 36 times faster can be achieved using the APS process relative to a conventional method. Using APS replica molding, biocompatible replicates from human fingerprints and gecko skin are fabricated using only a 3 min precursor solution loading step. Owing to the enhanced and reproducible resolution from APS replica molding, for the first time, the structural changes in the foot of a living gecko at the microscale can be observed when standing on a horizontal or vertical surface.

Host-microbial interactions between PTGR2 and Bifidobacterium in the early life gut of atopic dermatitis children.

BACKGROUND: Gut microbiota dysbiosis is linked to the development and responses of the immune system and can play an important role in the onset of allergic diseases including atopic dermatitis (AD). This study investigated the association between host genetics and the gut microbiota in AD. METHODS: A global gene expression profiling of the gut epithelial colonocytes, genetic variations analysis, and the gut microbial composition analysis were performed. RESULTS: This study identified the upregulation of PTGR2 (p = .028), a gene involved in prostaglandin catalysis and inflammatory responses, as a potential risk factor for AD. In subsequent fine mapping analysis using 17 single nucleotide polymorphisms (SNPs) of PTGR2 in 864 Korean subjects (420 AD patients and 444 unaffected controls), several SNPs and haplotypes showed significant associations with AD and its SCORing AD (SCORAD) values (p = .002). To investigate host-microbial interactions, further gut microbiota data and genotypes were obtained from an independent cohort of 176 subjects (91 AD patients and 85 controls). From correlation analysis, a significantly negative association between SNP and Bifidobacterium abundance was observed in AD patients (p = .005). In additional observations of PTGR2-associated downstream molecules, NRF2 (p = .004) and several antioxidant genes (GSTT1, GCLC, GPX1; p < .05) showed significantly reduced expression in AD patients. CONCLUSIONS: Our current findings suggest that the interaction between PTGR2 dysregulated expression and a Bifidobacterium abundance affects a higher risk of AD and a more severe onset.

Food allergy in early childhood increases the risk of oral allergy syndrome in schoolchildren: A birth cohort study.

BACKGROUND: The level of pollen in Korea has increased over recent decades. Research suggests that oral allergy syndrome (OAS) may be more frequent in childhood than previously recognized. We aimed to investigate the prevalence and characteristics of OAS in children aged 6-10 years from a general-population-based birth cohort. METHODS: We analyzed 930 children from the cohort for childhood origin of asthma and allergic diseases (COCOA). Allergic diseases were diagnosed annually by pediatric allergists. The skin prick tests were performed with 14 common inhalant allergens and four food allergens for the general population of children aged 3 and 7 years. RESULTS: Of the 930 eligible children, 44 (4.7%) aged 6-10 years were diagnosed with OAS. The mean age at onset was 6.74 years. OAS prevalence was 7.2% among children with allergic rhinitis (AR) and 19.1% among those with pollinosis, depending on comorbidity. OAS was more prevalent in schoolchildren with atopic dermatitis, food allergy, and sensitization to food allergens and grass pollen in early childhood. In schoolchildren with AR, only a history of food allergy until the age of 3 years increased the risk of OAS (aOR 2.971, 95% CI: 1.159-7.615). CONCLUSION: Food allergy and food sensitization in early childhood were associated with OAS in schoolchildren with AR. Further study is required to elucidate the mechanism by which food allergy in early childhood affects the development of OAS.

First report of post-harvest tuber rot of Apios americana Medikus caused by Penicillium expansum in South Korea.

Apios americana Medikus, a perennial vine legume native to North America, is known as 'Indian potato' or 'Apios' for their underground tubers that are used for human consumption in Korea (Choi et al., 2017). These tubers are known to be rich in isoflavones as well as other secondary products to have known several medicinal properties (Chu et al., 2019). The harvested tubers in 2020 were observed to rot during storage of tubers for 4 months at 4°C in the genetic resource storage room at the Bioenergy Crop Research Institute, National Institute of Crop Science, Muan, Jeollanam-do, South Korea. The incidence of rot symptoms with blue mold was less than 1% per 20 kg box. Ten infected tubers were collected from several boxes and the lesions were cut into small pieces, and then surface sterilized in 1.5% NaClO for 2 minutes, followed by rinsing 3 times with sterilized water. To investigate their morphological characteristics, ten isolates were cultured in Malt Extract Agar (MEA) medium at 25° C for 5 days (Pitt and Hocking, 1988). The surface morphology of the mycelium had white or light green fluffy, and completely blue spores were formed after about 5 days. The conidia were one-stage branched with an elliptical shape, about 3.5 to 4.3 × 2.9 to 3.6 (mean 3.8 ± 0.3 × 3.2 ± 0.2) ㎛ in diameter (n=30). Genomic DNAs of the isolates were extracted using Solgent DNA Extraction Kit (Solgent, Daejeon, Korea), and then PCR products of the internal transcribed spacer (ITS1/ITS4) region and the beta-tubulin gene (Bt2a/Bt2b) were sequenced and analyzed (Glass and Donaldson, 1995). The BLASTn showed that the representative isolate had 99% homology with reference Penicillium expansum strain ICMP 2708 (ITS region and TUB2 gene) in NCBI GenBank. The sequences of the isolate were deposited in GenBank as accession numbers MZ636667 and MZ702813 for ITS and TUB2 genes, respectively. Based on the morphological characteristics and molecular analysis, the isolate was identified as P. expansum. Pathogenicity assays of the isolate were also performed using three tubers in three replicates inoculated with spore suspension (concentration, 1×106 conidia/mL) and compared with a control group inoculated with sterilized water. The inoculated Apios tubers were placed in a plastic box maintained in conditions of high humidity at 25°C. Five days after inoculation, the typical symptoms were observed on inoculated tubers, and no symptoms were observed in the control one. P. expansum was again isolated from artificially inoculated tubers to complete Koch's assumption. This is the first report of P. expansum causing tuber rot in A. americana in South Korea. As the cultivated area of Apios is increasing in Korea, it will be necessary to develop effective storage methods and management strategies for the control of storage diseases such as blue mold.

First Report of Post-Harvest Tuber Rot of American Groundnut (Apios americana) Caused by Fusarium acuminatum.

Apios americana Medik, commonly known as American groundnut, is a leguminous perennial vine crop native to North America and is cultivated in Japan and Korea (Chu et al. 2019). Its tubers are edible and believed to be very nutritious, especially for women just after childbirth. The tubers also contain secondary metabolites, saponin and genistein, which is good for human health (Ichige et al. 2013). However, the storage of tubers at inappropriate temperatures and humidity levels can cause severe fungal infection, and adversely affect tuber quality. During March and April 2020, a white to pale-orange fungal mycelia were observed on stored American groundnut tubers, with 10 to 15% of seed tubers rotten. Infected tubers were collected, and fungal isolates were isolated on potato dextrose agar (PDA) using the single spore isolation method (Leslie and Summerell 2006). A pure culture (isolate JC20003) was obtained and stored at the Bioenergy Crop Research Institute, NICS, Muan, Republic of Korea. The fungus was cultured on PDA and V8 liquid media for 7 days at 25℃ to observe its morphological characteristics. The length and width of macroconidia ranged from 20.6 to 52.9 µm and 2.9 to 5.1 µm, respectively (n = 30). The microconidia were 8.5 to 14.9 µm and 2.3 to 4.2 µm in length and width, respectively (n = 30). Macroconidia were broadly falcate, strongly septate, 2 to 6 septations with dorsiventral curvature; chlamydospores were formed in chains; and microconidia were fusiform with 0 to 1 septation observed. Genomic DNA of the isolate was extracted using Solgent DNA extraction kit (Solgent, Daejeon, Korea), followed by PCR analysis using the internal transcribed spacer (ITS5/ITS4) and elongation factor (EF-1/EF2) genes (White et al. 1990; O'Donnel 2000). PCR products were sequenced and analyzed to confirm species identity (Yang et al. 2018). These sequences were deposited in GenBank (accession numbers MT703859/ITS and MT731939/EF). BLASTn search analysis showed 100% sequence similarity with Fusarium acuminatum (isolates N-51-1/ITS and WXWH24/EF). Based on morphological and molecular data analysis, the fungus was identified as F. acuminatum (Leslie and Summerell 2006; Marin et al. 2012). Pathogenicity tests were conducted on five tubers inoculated with 5 mm mycelial plugs with three replicates, while a non-mycelial plug served as the control. After 5 days of incubation in plastic containers at 25 °C with high humidity, typical symptoms developed. No symptoms were observed on the control tubers; F. acuminatum was re-isolated from artificially inoculated tubers to complete Koch's postulates. This is the first report on post-harvest tuber rot caused by F. acuminatum in Apios americana.

Functional Characterization of Two Cellulase Genes in the Gram-Positive Pathogenic Bacterium Clavibacter michiganensis for Wilting in Tomato.

Diverse plant pathogens secrete cellulases to degrade plant cell walls. Previously, the plasmid-borne cellulase gene celA was shown to be important for the virulence of the gram-positive bacterium Clavibacter michiganensis in tomato. However, details of the contribution of cellulases to the development of wilting in tomato have not been well-determined. To better understand the contribution of cellulases to the virulence of C. michiganensis in tomato, a mutant lacking cellulase activity was generated and complemented with truncated forms of certain cellulase genes, and virulence of those strain was examined. A celA mutant of the C. michiganensis type strain LMG7333 lost its cellulase activity and almost all its ability to cause wilting in tomato. The cellulase catalytic domain and cellulose-binding domain of CelA together were sufficient for both cellulase activity and the development of wilting in tomato. However, the expansin domain did not affect virulence or cellulase activity. The celA ortholog of Clavibacter sepedonicus restored the full virulence of the celA mutant of C. michiganensis. Another cellulase gene, celB, located in the chromosome, carries a single-base deletion in most C. michiganensis strains but does not carry a functional signal peptide in its N terminus. Nevertheless, an experimentally modified CelB protein with a CelA signal peptide was secreted and able to cause wilting in tomato. These results indicate that cellulases are major virulence factors of C. michiganensis that causes wilting in tomato. Furthermore, there are natural variations among cellulase genes directly affecting their function.

Skeletal Muscle Tissue Trib3 Links Obesity with Insulin Resistance by Autophagic Degradation of AKT2.

BACKGROUND/AIMS: Obesity is a serious health risk factor strongly associated with insulin resistance and type 2 diabetes; however, the underlying mechanisms associating obesity with insulin resistance remain unknown. In this study, we explored the physiological role of Trib3 in regulating glucose metabolism in skeletal muscle tissues in a Trib3 transgenic mice model. METHODS: Glucose metabolism in transgenic mice overexpressing Trib3 specifically in the skeletal muscle was examined by glucose/insulin tolerance test, metabolic cage studies, and glucose uptake assay. The effect of Trib3 overexpression on AKT phosphorylation and AKT protein turnover were assessed by RT-PCR and immunoblot analysis. Subcellular distribution of Trib3 and AKT1/2 was determined by microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. Ubiquitin assay was performed and ATG7 deficient cell line was employed to address the mechanisms of Trib3-dependent AKT protein homeostasis. RESULTS: We found that Trib3 expression in skeletal muscle is elevated in obese conditions, and transgenic mice that overexpressed Trib3, specifically in skeletal muscle tissues, displayed impaired glucose homeostasis by suppressing insulin-stimulated glucose uptake. Disruption of insulin signaling in skeletal muscle Trib3 transgenic mice may occur due to the specific downregulation of AKT2 but not AKT1. Autophagy regulated AKT2 protein turnover, and Trib3 overexpression stimulated autophagic degradation of AKT2 by promoting AKT2 ubiquitination. CONCLUSION: Because diet-induced obesity upregulates Trib3 and downregulates AKT2 in skeletal muscle tissues, Trib3 may play a key role in establishing an association between obesity and insulin resistance by regulating AKT2 protein homeostasis.

Multiple plasmid-borne virulence genes of Clavibacter michiganensis ssp. capsici critical for disease development in pepper.

Clavibacter michiganensis ssp. capsici is a Gram-positive plant-pathogenic bacterium causing bacterial canker disease in pepper. Virulence genes and mechanisms of C. michiganensis ssp. capsici in pepper have not yet been studied. To identify virulence genes of C. michiganensis ssp. capsici, comparative genome analyses with C. michiganensis ssp. capsici and its related C. michiganensis subspecies, and functional analysis of its putative virulence genes during infection were performed. The C. michiganensis ssp. capsici type strain PF008 carries one chromosome (3.056 Mb) and two plasmids (39 kb pCM1Cmc and 145 kb pCM2Cmc ). The genome analyses showed that this bacterium lacks a chromosomal pathogenicity island and celA gene that are important for disease development by C. michiganensis ssp. michiganensis in tomato, but carries most putative virulence genes in both plasmids. Virulence of pCM1Cmc -cured C. michiganensis ssp. capsici was greatly reduced compared with the wild-type strain in pepper. The complementation analysis with pCM1Cmc -located putative virulence genes showed that at least five genes, chpE, chpG, ppaA1, ppaB1 and pelA1, encoding serine proteases or pectate lyase contribute to disease development in pepper. In conclusion, C. michiganensis ssp. capsici has a unique genome structure, and its multiple plasmid-borne genes play critical roles in virulence in pepper, either separately or together.

Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

Clavibacter michiganensis subsp. capsici subsp. nov., causing bacterial canker disease in pepper.

Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies. One of these subspecies is C. michiganensis subsp. michiganensis, which causes bacterial canker disease in tomato. Bacterial strains showing very similar canker disease symptoms to those of a strain originally classified as C. michiganensis have been isolated from pepper. In this paper, we reclassified strains isolated from pepper. On the basis of phylogenetic analysis with 16S rRNA gene sequences, the strains isolated from pepper were grouped in a separate clade from other subspecies of C. michiganensis. Biochemical, physiological and genetic characteristics of strain PF008T, which is the representative strain of the isolates from pepper, were examined in this study. Based on multi-locus sequence typing and other biochemical and physiological features including colony color, utilization of carbon sources and enzyme activities, strain PF008T was categorically differentiated from eight subspecies of C. michiganensis. Moreover, genome analysis showed that the DNA G+C content of strain PF008T is 73.2 %. These results indicate that PF008T is distinct from other known subspecies of C. michiganensis. Therefore, we propose a novel subspecies, C. michiganensis subsp. capsici, causing bacterial canker disease in pepper, with a type strain of PF008T (=KACC 18448T=LMG 29047T).

Systematic review of the effects of mirror therapy in children with cerebral palsy.

[Purpose] To provide data for systematic intervention plans in occupational therapy practice by objectivity showing the value of mirror therapy interventions in children with cerebral palsy. [Subjects and Methods] Medline and EMBASE databases were searched for the key words "cerebral palsy," "mirror movement," "mirror therapy," and "mirror visual feedback." Nine studies that met the inclusion and exclusion criteria were identified. The qualitatively determined level of evidence, period of research, comparisons and interventions, tools used to measure the intervention, and the effects were analyzed. [Results] According to the results analyzed, one (1/9, 11.1%) study showed the same result as the control group, one (1/9, 11.1%) showed a negative effect, and seven (7/9, 77.8%) showed positive effects of mirror-mediated therapy, with meaningful improvement in function, such as hand strength, movement speed, muscle activity, and accuracy of hand matching. [Conclusion] Through this study, the value of mirror-mediated therapeutic interventions in occupational therapy practice targeting cerebral palsy was confirmed. It is expected that this result will be useful in establishing mirror therapy as an interventional program.

RAD001 (everolimus) enhances TRAIL cytotoxicity in human leukemic Jurkat T cells by upregulating DR5.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, is a promising new strategy for the treatment of cancer. However, aberrant PI3K/Akt/mTOR survival signaling may confer TRAIL resistance by altering the balance between pro- and anti-apoptotic proteins. In the present study, we showed that the Akt/mTOR inhibitor RAD001 (everolimus) induced cell death in a dose-dependent manner and enhanced TRAIL-induced apoptosis in human leukemic Jurkat T cells, which show PI3K/Akt/mTOR pathway activation and basal expression levels of death receptor (DR) 5 (TRAIL-R2). Investigation of the effect of RAD001 treatment on the expression of TRAIL receptors (TRAIL-Rs) in Jurkat T cells showed that RAD001 significantly upregulated DR5 by up to 51.22%, but not other TRAIL-Rs such as DR4 (TRAIL-R1), decoy receptor (DcR) 1 (TRAIL-R3), and DcR2 (TRAIL-R4). Pretreatment with DR5:Fc chimera abrogated the RAD001-induced increase of TRAIL cytotoxicity, indicating that the upregulation of DR5 by RAD001 plays a role in enhancing the susceptibility of Jurkat T cells to TRAIL. Our results indicate that combination treatment with RAD001 and TRAIL may be a novel therapeutic strategy in leukemia.

Molecular cloning, regulation, and functional analysis of two GHS-R genes in zebrafish.

Mammalian ghrelin is derived from stomach and regulates growth hormone release and appetite by modulating GHS-R (Growth hormone secretagogue receptor) activity. Zebrafish has been developed as a forward genetic screening model system and previous screening identified a number of genes involved in multiple signaling pathways. In this system, ghrelin has been identified and its function and regulation have been shown to be highly conserved to that of mammals. Here, we identified three isoforms of zGHS-R1 and one of zGHS-R2 (zGHS-R2a), and characterized their expression, regulation and function. Three isoforms of zGHS-R1, which we named zGHS-R1a, zGHS-R1b, and zGHS-R1c, are generated by alternative splicing. The expression of zGHS-R1 is highly enriched in brain, intestine tissue, and skin tissues. Compared to zGHS-R1, the expression pattern of zGHS-R2a is rather evenly distributed. A 15 day fasting elevated expression of zGHS-R1 and zGHS-R2 transcripts in anterior intestine tissues, but not in brain. Whereas zGHS-R1a, zGHS-R1c, and zGHS-R2a appear to be presented on the plasma membrane, the localization of zGHS-R1b seems to be restricted in the intracellular region. Treatment of ghrelin agonist, L692,585 or goldfish ghrelin peptides but not rat ghrelin, elevated intracellular Ca(2+) level and phosphorylation of ERK in HEK-293 cells expressing zGHS-R1a, but not zGHS-R1b, zGHS-R1c, or zGHS-R2a. It appears that besides core ghrelin peptide sequence of GS/TSF additional amino acids are required for the activation of zGHS-R1a, as rat ghrelin induces neither intracellular Ca(2+) mobilization nor ERK phosphrylation. These results suggest that ghrelin system in zebrafish is highly conserved to that of mammals, and thus is an ideal in vivo model for dissecting ghrelin system.