Innate-like Cytotoxic Function of Bystander-Activated CD8+ T Cells Is Associated with Liver Injury in Acute Hepatitis A.

Acute hepatitis A (AHA) involves severe CD8+ T cell-mediated liver injury. Here we showed during AHA, CD8+ T cells specific to unrelated viruses became activated. Hepatitis A virus (HAV)-infected cells produced IL-15 that induced T cell receptor (TCR)-independent activation of memory CD8+ T cells. TCR-independent activation of non-HAV-specific CD8+ T cells were detected in patients, as indicated by NKG2D upregulation, a marker of TCR-independent T cell activation by IL-15. CD8+ T cells derived from AHA patients exerted innate-like cytotoxicity triggered by activating receptors NKG2D and NKp30 without TCR engagement. We demonstrated that the severity of liver injury in AHA patients correlated with the activation of HAV-unrelated virus-specific CD8+ T cells and the innate-like cytolytic activity of CD8+ T cells, but not the activation of HAV-specific T cells. Thus, host injury in AHA is associated with innate-like cytotoxicity of bystander-activated CD8+ T cells, a result with implications for acute viral diseases.

Epigenetic scars in regulatory T cells are retained after successful treatment of chronic hepatitis C with direct-acting antivirals.

BACKGROUND & AIMS: Chronic HCV infection results in abnormal immunological alterations, which are not fully normalized after viral elimination by direct-acting antiviral (DAA) treatment. Herein, we longitudinally examined phenotypic, transcriptomic, and epigenetic alterations in peripheral blood regulatory T (Treg) cells from patients with chronic HCV infection before, during, and after DAA treatment. METHODS: Patients with chronic genotype 1b HCV infection who achieved sustained virologic response by DAA treatment and age-matched healthy donors were recruited. Phenotypic characteristics of Treg cells were investigated through flow cytometry analysis. Moreover, the transcriptomic and epigenetic landscapes of Treg cells were analyzed using RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin with sequencing) analysis. RESULTS: The Treg cell population - especially the activated Treg cell subpopulation - was expanded in peripheral blood during chronic HCV infection, and this expansion was sustained even after viral clearance. RNA sequencing analysis revealed that viral clearance did not abrogate the inflammatory features of these Treg cells, such as Treg activation and TNF signaling. Moreover, ATAC-seq analysis showed inflammatory imprinting in the epigenetic landscape of Treg cells from patients, which remained after treatment. These findings were further confirmed by intracellular cytokine staining, demonstrating that Treg cells exhibited inflammatory features and TNF production in chronic HCV infection that were maintained after viral clearance. CONCLUSIONS: Overall, our results showed that during chronic HCV infection, the expanded Treg cell population acquired inflammatory features at phenotypic, transcriptomic, and epigenetic levels, which were maintained even after successful viral elimination by DAA treatment. Further studies are warranted to examine the clinical significance of sustained inflammatory features in the Treg cell population after recovery from chronic HCV infection. IMPACT AND IMPLICATIONS: During chronic HCV infection, several immune components are altered both quantitatively and qualitatively. The recent introduction of direct-acting antivirals has led to high cure rates. Nevertheless, we have demonstrated that inflammatory features of Treg cells are maintained at phenotypic, transcriptomic, and epigenetic levels even after successful DAA treatment. Further in-depth studies are required to investigate the long-term clinical outcomes of patients who have recovered from chronic HCV infection.

Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization.

BACKGROUND: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. METHODS: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. RESULTS: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. CONCLUSIONS: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.

Diagnostic accuracy of non-invasive tests to screen for at-risk MASH-An individual participant data meta-analysis.

BACKGROUND & AIMS: There is a need to reduce the screen failure rate (SFR) in metabolic dysfunction-associated steatohepatitis (MASH) clinical trials (MASH+F2-3; MASH+F4) and identify people with high-risk MASH (MASH+F2-4) in clinical practice. We aimed to evaluate non-invasive tests (NITs) screening approaches for these target conditions. METHODS: This was an individual participant data meta-analysis for the performance of NITs against liver biopsy for MASH+F2-4, MASH+F2-3 and MASH+F4. Index tests were the FibroScan-AST (FAST) score, liver stiffness measured using vibration-controlled transient elastography (LSM-VCTE), the fibrosis-4 score (FIB-4) and the NAFLD fibrosis score (NFS). Area under the receiver operating characteristics curve (AUROC) and thresholds including those that achieved 34% SFR were reported. RESULTS: We included 2281 unique cases. The prevalence of MASH+F2-4, MASH+F2-3 and MASH+F4 was 31%, 24% and 7%, respectively. Area under the receiver operating characteristics curves for MASH+F2-4 were .78, .75, .68 and .57 for FAST, LSM-VCTE, FIB-4 and NFS. Area under the receiver operating characteristics curves for MASH+F2-3 were .73, .67, .60, .58 for FAST, LSM-VCTE, FIB-4 and NFS. Area under the receiver operating characteristics curves for MASH+F4 were .79, .84, .81, .76 for FAST, LSM-VCTE, FIB-4 and NFS. The sequential combination of FIB-4 and LSM-VCTE for the detection of MASH+F2-3 with threshold of .7 and 3.48, and 5.9 and 20 kPa achieved SFR of 67% and sensitivity of 60%, detecting 15 true positive cases from a theoretical group of 100 participants at the prevalence of 24%. CONCLUSIONS: Sequential combinations of NITs do not compromise diagnostic performance and may reduce resource utilisation through the need of fewer LSM-VCTE examinations.

Correlation of LLT-1 and NLRC4 inflammasome and its effect on glioblastoma prognosis.

PURPOSE: LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. METHODS: GBM tissues were collected for RNA sequencing (RNA-seq) and Immunofluorescent experiments. Colocalization of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 to 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively. These data were subjected to survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single-cell RNA-seq analysis. RESULTS: In GBM tissues, LLT-1 was predominantly colocalized with glial fibrillary acidic protein (GFAP)-expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were mainly co-expressed in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells showed high NF, NLR, and IL-1 signaling pathway enrichment compared to other malignant cell types. CONCLUSION: We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new NK-cell-mediated anti-glioma approaches.

Trends in alcohol use and alcoholic liver disease in South Korea: a nationwide cohort study.

BACKGROUND: There is a lack of national-level research on alcohol consumption and the epidemiology of alcoholic liver disease (ALD) in South Korea. This study aims to address the critical public health issue of ALD by focusing on its trends, incidence, and outcomes, using nationwide claims data. METHODS: Utilizing National Health Insurance Service data from 2011 to 2017, we calculated the population's overall drinking amount and the incidence of ALD based on ICD-10 diagnosis codes. RESULTS: From 2011 to 2017 in South Korea, social drinking increased from 15.7% to 16.5%, notably rising among women. High-risk drinking remained around 16.4%, decreasing in men aged 20-39 but not decreased in men aged 40-59 and steadily increased in women aged 20-59. The prevalence of ALD in high-risk drinkers (0.97%) was significantly higher than in social drinkers (0.16%). A 3-year follow-up revealed ALD incidence of 1.90% for high-risk drinkers and 0.31% for social drinkers. Women high-risk drinkers had a higher ALD risk ratio (6.08) than men (4.18). The economic burden of ALD was substantial, leading to higher healthcare costs and increased hospitalization. Progression rates to liver cirrhosis and hepatocellular carcinoma (HCC) in ALD patients were 23.3% and 2.8%, respectively, with no gender difference in cirrhosis progression. CONCLUSIONS: The study revealed a concerning rise in alcohol consumption among South Korean women and emphasizes the heightened health risks and economic burdens associated with high-risk drinking, especially concerning ALD and its complications.

The Epidemiology of Hepatitis B Virus Infection in Korea: 15-Year Analysis.

BACKGROUND: The purpose of this study is to investigate the epidemiological changes in chronic hepatitis B (CHB) and assess the impact of coronavirus disease 2019 (COVID-19) over the past 15 years in a region endemic to hepatitis B virus (HBV). METHODS: National Health Insurance Service claims data of hepatitis B patients spanning from 2007 to 2021 was utilized. To compare the characteristics of the hepatitis B group, a control group adjusted for age and gender through propensity score matching analysis was established. RESULTS: The number of patients with CHB has consistently increased over the past 15 years. The average age of the CHB patient group has shown a yearly rise, while the prevalence of male dominance has gradually diminished. The proportions of hepatocellular carcinoma, liver cirrhosis, and decompensation have exhibited a declining pattern, whereas the proportion of liver transplants has continuously risen. Patients with CHB have demonstrated significantly higher medical and medication costs compared to the control group. Moreover, patients with CHB have shown a higher prevalence of comorbidities along with a significantly higher rate of concomitant medication usage. During the COVID period, the HBV group experienced a substantial decrease in the number of outpatient visits and overall medical costs compared to the control group. CONCLUSION: The epidemiology of CHB has undergone significant changes over the past 15 years, encompassing shifts in prevalence, severity, medical costs, and comorbidities. Furthermore, the impact of COVID-19 has been observed to decrease healthcare utilization among patients with CHB when compared to controls.

First Report of Anthracnose on Euonymus japonicus Thunb. Caused by a Provisionally Novel Species of Colletotrichum in the Magnum Complex in South Korea.

Euonymus japonicus Thunb., also known as the evergreen spindle tree, is an evergreen tree, which is widely planted as a hedge plant along streets in South Korea. In April 2022, severe anthracnose symptoms were observed on the leaves of this tree in Jangsu in the Jeonbuk Province of the country (35°43'49.44″N, 127°34'53.7″E). About 80% of the leaves of each affected tree within a 0.03-ha area showed incidence of the disease on approximately 30 trees were planted along the roadside (~30 m). These symptoms typically included circular or irregularly shaped whitish-gray lesions with a diameter of 2.0 to 3.0 cm. In cases where some leaves were severely affected, larger blotches formed. To isolate the pathogen, about ten leaves showing anthracnose symptoms on each tree were randomly selected and brought to the laboratory. Fungal isolations were made from acervuli filled with conidial masses on infected evergreen tissues, followed by plating onto 2% potato dextrose agar (PDA) as well as incubated at 25℃. On the PDA, colonies were circular, raised, green-grey or dark grey, and had a distinct white margin. The conidia were single-celled, transparent, cylindrical with rounded ends, had smooth walls, with a length ranging from 12 µm to 16.7 µm and a width raging from 4 µm to 6.5 µm (av. = 14.1 X 5.0 µm, n=40). Of those that were successfully recovered with approximately 90% frequency, two monoconidial isolates were deposited to the culture collection at Chungnam National University in South Korea (Accession number: CDH059-060). To ensure the identity of the fungus, genomic DNAs were extracted from the selected isolates, CDH059-060, and were sequenced. This was achieved based on partial sequences of the internal transcribed spacer (ITS), actin and beta-tubulin (TUB2) gene regions which were amplified using ITS1F / ITS4 (Gardes and Bruns 1993; White et al. 1990), ACT-512F / ACT-783R (Carbone and Kohn 1999), and T1 / Bt2b (O'Donnell and Cigelnik 1997; Glass and Donaldson 1995) primer pairs, respectively. The resulting sequences were deposited to GenBank (OR984424-425) for ITS, (OR996289-290) for actin, and (OR996291-292) for TUB2. For a phylogenetic analysis, sequences from different gene regions (ITS, actin and TUB2) retrieved from GenBank were aligned, concatenated, and analyzed as a single dataset based on a maximum likelihood analysis. The phylogenetic result revealed that the fungus isolated in this study was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Colletotrichum, which is most closely related to Colletotrichum liaoningense (Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, CGMCC3.17616 (KP890104 for ITS, KP890097 for actin, and KP890111 for TUB, Diao et al. 2017). Sequence comparisons revealed that this pathogen differed from C. liaoningense at 20 of 494 characters (∼4.0%) in the ITS and 2 of 251 (∼1.0%) in the actin sequences. For pathogenicity tests, three seedlings of E. japonicus were used. The leaves for each tree were treated with 10 ml of a conidial suspension by spraying (1x106 conidia ml-1 of the isolate, CDH059), while the three seedlings were treated with distilled water as control. After sprayed, the treated areas were sealed with plastic bags for a day to maintain humidity. Anthracnose symptoms identical to those observed in the field appeared seven days after inoculations, while no symptoms were observed in the control. Re-isolations were successfully achieved from the treatments, fulfilling Koch's postulates. Anthracnose associated with the provisionally novel species of Colletotrichum sp. on E. japonicus has not been recorded elsewhere, and in this regard, this is the first report of anthracnose caused by Colletotrichum sp. on E. japonicus in Korea. To effectively control the disease, more attention should be paid to the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 µm long and 3.1 to 4.5 µm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and ß-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

Long-Term Effect of PNPLA3 on the Aggravation of Nonalcoholic Fatty Liver Disease in a Biopsy-Proven Cohort.

The PNPLA3 rs738409 G allele increases the risk of not only nonalcoholic fatty liver disease (NAFLD) but also nonalcoholic steatohepatitis (NASH) or fibrosis.1 It also affects the prognosis of patients with NAFLD in specific conditions. After liver transplantation, patients with NAFLD carrying the rs738409 GG genotype have a higher risk of graft steatosis2 or development of hepatocellular carcinoma.3 In addition, rs738409 modifies the effects of medical intervention: patients with NAFLD carrying the GG genotype showed a lower effect of omega-3 polyunsaturated fatty acid treatment on the reduction of liver fat;4 in contrast, they were more sensitive to the beneficial effects of lifestyle modifications.5,6.

Myosteatosis, but not Sarcopenia, Predisposes NAFLD Subjects to Early Steatohepatitis and Fibrosis Progression.

BACKGROUND & AIMS: Sarcopenia and myosteatosis are associated with advanced nonalcoholic fatty liver disease (NAFLD). However, muscle alterations in early stage NAFLD remain unclear. METHODS: Patients with nonalcoholic fatty liver (NAFL) or early nonalcoholic steatohepatitis (NASH) without significant fibrosis were selected from a prospective biopsy-proven NAFLD cohort (N = 338). The skeletal muscle index and mean muscle attenuation (MA) were measured using abdominal fat computed tomography at the third lumbar vertebra level. Severe myosteatosis was defined as the lowest quartile of sex-stratified MA values. RESULTS: Patients with early NASH (n = 87) had lower MA (45.61 ± 6.45 vs 47.48 ± 5.85 HU; P = .028) than patients with NAFL (n = 251) but a similar skeletal muscle index. Patients with more severe lobular inflammation and hepatocellular ballooning had lower MA (P = .003 and P = .041, respectively). The severe myosteatosis prevalence was higher in early NASH than in NAFL (33.3% vs 21.1%; P = .029). Patients with severe myosteatosis were more likely to have early NASH in multivariable analysis adjusted for age, sex, and metabolic factors (odds ratio, 2.45; 95% confidence interval (CI), 1.24-4.86), which was maintained after adjustment for visceral fat amount (odds ratio, 2.44; 95% CI, 1.22-4.89). During a median 29-month follow-up, 170 patients underwent repeated transient elastography. Fibrosis progression-an increase in liver stiffness measurement >2 kPa or second liver stiffness measurement ≥7 kPa-was found in 28 and 31 individuals. Severe myosteatosis was significantly associated with fibrosis progression after adjustment for various confounders (hazard ratio, 2.49; 95% CI, 1.15-5.40 and hazard ratio, 2.09; 95% CI, 1.01-4.34 for different fibrosis progression definitions). CONCLUSIONS: Severe myosteatosis is significantly associated with early NASH and fibrosis progression in early stage NAFLD.

Micromonospora antibiotica sp. nov. and Micromonospora humidisoli sp. nov., two new actinobacterial species exhibiting antimicrobial potential.

Two novel actinobacterial strains, designated MMS20-R2-23T and MMS20-R2-29T, were isolated from riverside soil and subjected to taxonomic characterization. Both strains were Gram-stain-positive, aerobic, non-motile and filamentous, and formed orange to strong orange-brown coloured colonies, which later turned black. Both strains grew optimally at mesophilic temperatures, neutral to slightly alkaline pH and in the absence of NaCl. Analysis of 16S rRNA gene sequences indicated that the two novel strains fell into phylogenetic clusters belonging to the genus Micromonospora. Strains MMS20-R2-23T and MMS20-R2-29T showed the highest 16S rRNA gene sequence similarity to Micromonospora phytophila SG15T (99.3 %) and Micromonospora humida MMS20-R1-14T (99.4 %), respectively. Based on the comparative genome analysis, strain MMS20-R2-23T had the highest orthologous average nucleotide identity (orthoANI) value of 92.70 % with Micromonospora matsumotoense DSM 44100T, and MMS20-R2-29T shared 94.99 % with Micromonospora wenchangensis CCTCC AA 2012002T. Besides, the digital DNA-DNA hybridization (dDDH) values of MMS20-R2-23T and MMS20-R2-29T with the same species were 47.6 and 59.2% respectively, which were also highest among the compared species, thus confirming the separation of each strain at species level from related species. The orthoANI and dDDH values between MMS20-R2-23T and MMS20-R2-29T were 92.18 and 44.9% respectively. The genomes of strains MMS20-R2-23T and MMS20-R2-29T were estimated as 7.56 Mbp and 7.13 Mbp in size, and the DNA G+C contents were 72.5 and 72.9 mol%, respectively. The chemotaxonomic properties of both strains were consistent with those of the genus. The novel strains showed antimicrobial activity against a broad range of microbes, in particular Gram-positive bacteria and yeasts. It is evident that each of the isolated strains merits recognition as representing novel species of Micromonospora, for which the names Micromonospora antibiotica sp. nov. (type strain=MMS20-R2-23T=KCTC 49542T=JCM 34495T) and Micromonospora humidisoli sp. nov. (type strain=MMS20-R2-29T=KCTC 49543T=JCM 34496T) are proposed.

Pseudomonas insulae sp. nov., isolated from island soil.

A novel Gram-stain-negative, rod-shaped, aerobic and motile bacterium designated strain UL073T was isolated from a forest soil of an island, and subjected to taxonomic characterization. Strain UL073T grew at 10-37 °C (optimum, 30 °C), at pH 5.0-10.0 (optimum, pH 7.0) and in the presence of 0-3 % NaCl (optimum, 0 %), respectively. Strain UL073T showed the highest sequence similarity to Pseudomonas lalkuanensis PE08T based on 16S rRNA gene analysis with a sequence similarity of 98.08 %, which was well below the suggested cutoff for species distinction. The 16S rRNA gene tree as well as the multilocus sequence analysis and genome-based trees indicated the independent taxonomic position of strain UL073T, and the orthologous average nucleotide identity and in silico DNA-DNA hybridization values between strain UL073T and related species were no higher than 84.7 and 28.3% respectively, thus confirming the distinctive taxonomic position of the strain. The chemotaxonomic properties were consistent with those of the genus, as the major fatty acids of the strain were a summed feature consisting of C18 : 1 ω7c/C18 : 1 ω6c (31.4 %), another summed feature consisting of C16 : 1 ω7c/C16 : 1 ω6c (23.1 %), and C16 : 0 (22.0 %), the major respiratory quinone was ubiquinone 9, and the major polar lipids were phosphatidylethanolamine and diphosphatidylglycerol. The genome size and DNA G+C content of strain UL073T were 4.87 Mbp and 65.9 mol%. On the basis of phenotypic and phylogenetic evidence, strain UL073T should be classified as representing a novel species of Pseudomonas, for which the name Pseudomonas insulae sp. nov. (type strain=UL073T=KCTC 82407T=JCM 34511T) is proposed.

Paenibacillus artemisiicola sp. nov. and Paenibacillus lignilyticus sp. nov., two new endophytic bacterial species isolated from plant roots.

Two Gram-positive, endospore-forming, rod-shaped bacterial strains designated MWE-103T and DLE-14T were isolated from plant roots. The 16S rRNA gene sequence analysis indicated that strain MWE-103T was closely related to Paenibaillus sacheonensis SY01T with a sequence similarity of 97.82 %, and strain DLE-14T to Paenibacillus rhizoryzae IZS3-5T with 99.09 % similarity. The orthologous average nucleotide identity and digital DNA-DNA hybridization values using whole genome data indicated that strains MWE-103T and DLE-14T could be readily distinguished from the mostly related species. Both strains grew at mesophilic temperature ranges, and grew best at pH 6 and in the absence of NaCl. The major fatty acid in both strains was anteiso-C15 : 0, but their relative proportions differed. The predominant quinone of both strains was menaquinone 7, the cell-wall diamino acid was meso-diaminopimelic acid, and the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, which were consistent with those of related species. Amylase and cellulase activities were positive for both strains. Strain DLE-14T exhibited the potential for lignin degradation. The DNA G+C contents of strain MWE-103T and DLE-14T were 60.9 and 50.8 mol% respectively. The genomes of the two strains revealed potential plant-growth-promoting characteristics such as nitrogen fixation, siderophore production and phosphate solubilization. Based on phylogenetic and phenotypic evidence, strains MWE-103T and DLE-14T should each be recognized as a novel species of Paenibacillus, for which the names Paenibacillus artemisiicola sp. nov. (type strain: MWE-103T=KCTC 43287T=JCM 34503T) and Paenibacillus lignilyticus sp. nov. (type strain: DLE-14T=KCTC 43288T=JCM 34504T) are proposed.

Streptomyces montanisoli sp. nov., exhibiting antimicrobial activity, isolated from mountain soil around a decaying tree.

A Gram-stain-positive, aerobic actinobacterial strain designated MMS17-BM035T isolated from mountain soil around a decaying tree was subjected to taxonomic characterization. The isolate developed extensively branched substrate mycelia and white aerial hyphae on International Streptomyces Project 2 agar. Strain MMS17-BM035T grew at 15-34 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-6 % NaCl (optimum, 0 %). Analysis of 16S rRNA gene sequences indicated that MMS17-BM035T fell into a phylogenetic cluster belonging to the genus Streptomyces. MMS17-BM035T shared the highest sequence similarity of 99.45 % with Streptomyces fuscigenes JBL-20T, and no higher than 98.7 % with other species of Streptomyces. Based on the orthologous average nucleotide identity, MMS17-BM035T was again mostly related to S. fuscigenes JBL-20T with 84.14 % identity, and less than 80 % with other species. The digital DNA-DNA hybridization analysis also indicated low levels of relatedness with related species, as the highest value was observed with S. fuscigenes JBL-20T (28.8 %). The major fatty acids of the strain were anteiso-C15 : 0, a summed feature (consisting of C18 : 1 ω7c/C18 : 1 ω6c), iso-C15 : 0, C16 : 0 and C20 : 0. The major respiratory quinones were MK-9(H4) and MK-9(H6). The diagnostic polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositolmannoside. The major cell-wall diamino acid was ll-diaminopimelic acid, and the characteristic whole-cell sugars were glucose and ribose. The DNA G+C content was 72.1 mol%. Strain MMS17-BM035T exhibited antimicrobial activity against several Gram-positive bacteria and yeasts. Based on both phenotypic and phylogenetic evidences, strain MMS17-BM035T should be classified as representing a novel species, for which the name Streptomyces montanisoli sp. nov. (type strain=MMS17-BM035T=KCTC 49544T=JCM 34528T) is proposed.

Efficacy and Safety of Pregabalin for Muscle Cramps in Liver Cirrhosis: A Double-Blind Randomized Controlled Trial.

BACKGROUND: Muscle cramp is possibly related to peripheral nerve hyperexcitability (PNH), and one of the most debilitating symptoms frequently encountered in patients with liver cirrhosis. We investigated whether pregabalin, a gamma-aminobutyric acid analogue, can suppress neuronal excitability and reduce muscle cramps in cirrhotic patients. METHODS: We conducted a randomized, double-blind, placebo-controlled trial in which study participants with cirrhosis from a single tertiary center were enrolled. Primary endpoint was the relative change in cramp frequency from the run-in to standard dose treatment phase (4 weeks per each). Secondary endpoints included the responder rate, and the changes in cramp frequency during sleep, pain intensity, health-related quality of life (Liver Disease Quality of Life Instrument, Short Form-36) and electrophysiological measures of PNH. RESULTS: This study was terminated early because of insufficient accrual. 80% (n = 56) of the target number of participants (n = 70) were randomized to pregabalin (n = 29) or placebo (n = 27). Median baseline frequency of muscle cramps (interquartile range) was 5.8 (3.5-10) per week in the pregabalin group and 6.5 (4.0-10) in the placebo group (P = 0.970). The primary analysis showed a significant reduction in cramp frequency with pregabalin compared to placebo (-36% vs. 4.5% for the percentage change, P = 0.010). Secondary outcomes did not differ significantly between the two groups. Adverse effects with pregabalin were mainly dizziness and lethargy. CONCLUSION: With multiple problems emerging from premature termination in mind, the results suggested an acceptable safety profile and favorable effect of pregabalin in reducing muscle cramps compared to placebo in cirrhotic patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01271660.

First Report of Anthracnose on Juglans regia Caused by Colletotrichum siamense in Korea.

Walnut (Juglans regia) is one of the main tree crops cultivated for nut production in South Korea with an estimated production of about 1,189 tons per year (Korea Forest Service 2020). In August 2021, anthracnose symptoms, including dark, depressed, irregularly shaped lesions on fruits and leaves of walnut cv. Sinlyeong, were observed at three orchards in Nonsan (36°10'22.5"N 127°06'14.8"E) and Suwon (37°16'04.7"N 126°55'22.3"E and 37°15'10.6"N 126°57'35.6"E). This led to severe yield loss of walnut fruit with a disease incidence of approximately 70 to 80% in each orchard. Three samples, including infected fruits and leaves, were randomly collected per site. Fungal isolates were isolated either from acervuli filled with conidial masses on infected walnut tissues or from plant tissues that were surface-disinfested, followed by plating onto 2% PDA. Colonies were initially white, later became pale brownish to light gray with concentric rings of salmon sporodochia. White to gray aerial mycelia, reaching 65 mm diameter in 5 days, were abundantly produced on PDA at 25 °C. Appressoria were brown, ovoid, and in some cases, clavate, 5.1-8.7 µm in length, and 3.2-5.1 µm in width (n = 50). Conidia were single celled, hyaline, cylindrical with rounded ends and smooth walls, guttulate, 13.6-18.8 µm in length, and 4.4-6.3 µm in width (n = 50). Setae were absent. Three isolates, i.e., one per orchard, were retained and deposited in the culture collection (CDH) of National Institute of Forest Science, Korea (Accession No. CDH052-054). The internal transcribed spacer (ITS) region of rDNA, beta-tubulin (TUB2) and a partial sequence of the actin (ACT) genes were amplified and sequenced for each of the isolates using the pair of primers, ITS1F/ITS4 (Gardes and Bruns 1993; White et al. 1990), T1/Bt2b (ODonnell and Cigelnik 1997; Glass and Donaldson 1995) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. A BLAST search in GenBank revealed that the sequences of ITS (OK631731-733), TUB2 (OK665927-929) and ACT (OK665930-932) showed sequence identities of 98.6 to 99.6% to Colletotrichum siamense sequences (FJ972613, FJ907423, FJ907438). A maximum likelihood tree, based on a combined dataset of ITS, ACT and TUB2 gene sequences for Colletotrichum spp., revealed that the three isolates were clustered with type specimens of C. siamense. To prepare larger quantities of inoculum for the pathogenicity, mycelial plugs bearing acervuli taken from 2% PDA were incubated in a conical flask containing 200 ml of 2% potato dextrose broth at 25°C on a rotary shaker at 150 rpm for two weeks. Spore concentration was adjusted to 1.0 × 104 ml-1 conidia of C. siamense (CDH054). A 10 to 15 ml of spore suspension was then sprayed on each leaf of 12 seedlings of 'Sinlyeong' walnut (three-year-old), while 7 seedlings were treated with sterile distilled water as a control. Each treated seedling was covered by a plastic bag to maintain moisture for one day. Inoculation trials were repeated twice, in August and September 2021. Symptoms identical to those observed in the field developed four to five days after the inoculations from which the inoculated pathogen was successfully re-isolated, fulfilling Koch's postulates. However, no symptoms were observed in the control. To our knowledge, this is the first report of anthracnose on J. regia caused by C. siamense in Korea. This indicates that disease occurrences must be further rigorously surveyed at the nation-wide scale to effectively control the disease in the country.

Latissimus dorsi transfer vs. lower trapezius transfer for posterosuperior irreparable rotator cuff tears.

BACKGROUND: Latissimus dorsi transfer (LDT) and lower trapezius transfer (LTT) are effective treatment options for posterosuperior irreparable rotator cuff tears (IRCTs) in relatively young patients and elderly high-demand patients without arthritic changes. However, the optimal treatment option for patients with posterosuperior IRCT remains a subject of ongoing debate. This study aimed to compare clinical and radiologic short-term outcomes between arthroscopic-assisted LDT (aLDT) and arthroscopic-assisted LTT (aLTT) in patients with posterosuperior IRCT. METHODS: This retrospective crossover study included patients who underwent aLDT or aLTT for posterosuperior IRCT and who had a minimum clinical follow-up time of 2 years after undergoing surgical procedures between January 2012 and June 2019. A total of 90 patients with posterosuperior IRCT were divided into 2 groups according to the surgical procedure: group D underwent aLDT (n = 48) and group T underwent aLTT (n = 42). Clinical outcomes comprised the visual analog scale score for pain, active shoulder range of motion (ROM), American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES) score, and activities of daily living that require active external rotation (ADLER) score. Radiologic outcomes included acromiohumeral distance (AHD). The progression of arthritis was evaluated using Hamada grade. Graft integrity was assessed using postoperative magnetic resonance imaging. RESULTS: Significant improvements in clinical outcomes were observed in both groups. Active shoulder external rotation (P < .001), postoperative ASES score (P < .001), and ADLER score (P < .001) were significantly higher in group T than in group D. AHD at 2-year follow-up was significantly higher in group T than in group D (P < .001). The rate of progression of arthritis was significantly higher in group D (31.3%) than in group T (7.1%) (P = .031). CONCLUSIONS: Although both LDT and LTT improved the overall clinical outcomes of patients with posterosuperior IRCT, LTT was superior to LDT in terms of shoulder ROM, functional improvement, and progression of arthritis. Our findings indicate that LTT may be the preferred treatment option for posterosuperior IRCT in relatively active and young patients.

Association between circulating bile acid alterations and nonalcoholic steatohepatitis independent of obesity and diabetes mellitus.

BACKGROUND AND AIMS: Bile acid (BA) dysregulation is related to not only metabolic diseases but also nonalcoholic fatty liver disease (NAFLD). We investigated whether circulating BA levels are altered according to the histological severity of NAFLD independent of metabolic derangements. METHODS: Global metabolic profiling and targeted BA analysis using sera collected from biopsy-proven no-NAFLD (n = 67), nonalcoholic fatty liver (NAFL) (n = 99), and nonalcoholic steatohepatitis (NASH, n = 75) subjects were performed sequentially. Circulating metabolome analysis integrated with the hepatic transcriptome was performed to elucidate the mechanistic basis of altered circulating BA profiles after stratification by obesity (body mass index ≤ 25 kg/m2 ). Circulating BA alterations were also validated in an independent validation cohort (29 no-NAFLD, 70 NAFL and 37 NASH). RESULTS: Global profiling analysis showed that BA was the metabolite significantly altered in NASH compared to NAFL. Targeted BA analysis demonstrated that glyco-/tauro-conjugated primary BAs were commonly increased in nonobese and obese NASH, while unconjugated primary BAs increased only in nonobese NASH. These characteristic primary BA level changes were maintained even after stratification according to diabetes status and were replicated in the independent validation cohort. Compared to nonobese NAFL patients, nonobese NASH patients exhibited upregulated hepatic expression of CYP8B1. CONCLUSIONS: BA metabolism is dysregulated as the histological severity of NAFLD worsens, independent of obesity and diabetes status; dysregulation is more prominent in nonobese NAFLD patients. Metabolome-driven omics approach provides new insight into our understanding of altered BA metabolism associated with individual phenotypes of NAFLD.

Pseudomonas guryensis sp. nov. and Pseudomonas ullengensis sp. nov., isolated from soil.

Two Gram-staining-negative, aerobic, rod-shaped bacteria designated strains SR9T and UL070T, were isolated from soil and subjected to taxonomic characterization. Strain SR9T grew at 10-37 °C (optimum 30 °C), at pH 4.0-10.0 (optimum pH 8.0) and in the presence of 0-1 % NaCl (optimum 0 %), and UL070T at 4-33 °C (optimum 30 °C), at pH 4.0-10.0 (optimum pH 7.0) and in the presence of 0-2 % NaCl (optimum 0 %), respectively. Strain UL070T was motile with flagella. Analysis of 16S rRNA gene sequences indicated that the two strains fell into phylogenetic clusters belonging to the genus Pseudomonas. Both strains SR9T and UL070T were mostly related to Pseudomonas campi S1-A32-2T with 99.70 and 99.01% sequence similarities, and the similarity between the two isolates was 98.90 %. The genome-based in silico analyses indicated that each of the strains SR9T and UL070T was clearly separated from other species of Pseudomonas, as the orthologous average nucleotide identity (OrthoANI) and the digital DNA-DNA hybridization (dDDH) values were no higher than 93.09 and 50.03% respectively with any related species, which were clearly below the cutoff for species distinction. The fatty acid profiles of the two strains mainly consisting of unsaturated components, the presence of ubiquinone 9 (Q-9) as the major respiratory quinone, and phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) as the diagnostic polar lipids were consistent with their classification into Pseudomonas. The DNA G+C contents of strains SR9T and UL070T were 63.2 mol% and 63.6 mol% respectively. On the basis of both phenotypic and phylogenetic evidences, each of the isolated strains should be classified as a novel species, for which the names Pseudomonas guryensis sp. nov. (type strain=SR9T=KCTC 82228T=JCM 34509T) and Pseudomonas ullengensis sp. nov. (type strain=UL070T=KCTC 82229T=JCM 34510T) are proposed.