Cloning and Expression Analysis of Bioluminescence Genes in Omphalotus guepiniiformis Reveal Stress-Dependent Regulation of Bioluminescence.

Bioluminescence is a type of chemiluminescence that arises from a luciferase-catalyzed oxidation reaction of luciferin. Molecular biology and comparative genomics have recently elucidated the genes involved in fungal bioluminescence and the evolutionary history of their clusters. However, most studies on fungal bioluminescence have been limited to observing the changes in light intensity under various conditions. To understand the molecular basis of bioluminescent responses in Omphalotus guepiniiformis under different environmental conditions, we cloned and sequenced the genes of hispidin synthase, hispidin-3-hydroxylase, and luciferase enzymes, which are pivotal in the fungal bioluminescence pathway. Each gene showed high sequence similarity to that of other luminous fungal species. Furthermore, we investigated their transcriptional changes in response to abiotic stresses. Wound stress enhanced the bioluminescence intensity by increasing the expression of bioluminescence pathway genes, while temperature stress suppressed the bioluminescence intensity via the non-transcriptional pathway. Our data suggested that O. guepiniiformis regulates bioluminescence to respond differentially to specific environmental stresses. To our knowledge, this is the first study on fungal bioluminescence at the gene expression level. Further studies are required to address the biological and ecological meaning of different bioluminescence responses in changing environments, and O. quepiniiformis could be a potential model species.

Flavivirga spongiicola sp. nov. and Flavivirga abyssicola sp. nov., Isolated from Marine Environments.

Two novel Gram-stain-negative, strictly-aerobic, rod-shaped (1.2 ± 3.4 µm × 0.3 ± 0.7 µm), and non-motile marine bacterial species, designated MEBiC05379T and MEBiC07777T, were isolated from a marine sponge Pseudaxinella sp. in Gangneung City and deep-sea sediments of the Ulleung basin in the East Sea of Korea, respectively. The 16S rRNA gene sequence analysis revealed high levels of similarities between these strains and members of the genus Flavivirga (97.0-98.4% sequence identities). Both novel strains revealed as mesophilic, neutrophilic in pH and slightly halophilic. Similar to those of other Flavivirga members, the primary cellular fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C15:03-OH, and iso-C17:0 3-OH, with MEBiC05379T and MEBiC07777T containing relatively higher proportions of C12:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). In both taxa, the major isoprenoid quinone was MK-6. The DNA G + C contents of MEBiC05379T and MEBiC07777T genomes were 32.62 and 32.46 mol%, respectively. Compared to other members of Flavivirga, both strains exhibited similar DNA G + C ratio and fatty acids pattern, yet enzyme expression and carbon sources utilization pattern were different. Genomes of the genus Flavivirga showed enzyme preferences to fucoidan and sulfated galactans. Considering the monophyly rule, AAI values delineate the genus Flavivirga from adjacent genera calculated to be 76.0-78.7%. Based on the phenotypic, genomic and biochemical data, strains for MEBiC05379T and MEBiC07777T thus represent two novel species in the genus Flavivirga, for which the names Flavivirga spongiicola sp. nov. (MEBiC05379T [= KCTC 92527 T = JCM 16662 T]), and Flavivirga abyssicola sp. nov. (MEBiC07777T [= KCTC 92563 T = JCM 36477 T]) are proposed.

AZD7648, a DNA-PKcs inhibitor, overcomes radioresistance in Hep3B xenografts and cells under tumor hypoxia.

Radiation therapy is one of the most commonly used cancer treatments. However, it has important concerns such as damage to normal tissues around cancers and radioresistance. To overcome these problems, combination therapy using radiosensitizer and radiotherapy will be a good alternative. The present study investigated the effects of AZD7648 on overcoming radioresistance as well as radiosensitizing in Hep3B xenografts and cells. The results showed that AZD7648 enhanced ionizing radiation (IR)-induced tumor growth not only in radiosensitive but also radioresistant tumors. In particular, the combination of AZD7648 with radiation reduced the expression of hypoxia induce factor-1α (HIF-1α) in radioresistant tumors. In vitro studies, AZD7648 plus IR increased IR-induced G2/M arrest and regulated cell cycle checkpoints such as cyclinB1, p-cdc2 in normoxia but not in hypoxia. AZD7648 induced more radiation-mediated ROS than radiation only under normoxia, but these ROS were not altered by AZD7648 under hypoxia. Interestingly, AZD7648 downregulated HIF-1α expression level under CoCl2-treated hypoxic condition but not in normoxic condition. In conclusion, AZD7648 synergistically increased radiosensitivity through accumulating IR-induced G2/M arrest and further improved radioresistance via regulation of HIF-1α. The present data suggest that AZD7648 may be a strong radiosensitizer in radioresistant as well as radiosensitive cancers.

Genomic potential and physiological characteristics of C1 metabolism in novel acetogenic bacteria.

Acetogenic bacteria can utilize C1 compounds, such as carbon monoxide (CO), formate, and methanol, via the Wood-Ljungdahl pathway (WLP) to produce biofuels and biochemicals. Two novel acetogenic bacteria of the family Eubacteriaceae ES2 and ES3 were isolated from Eulsukdo, a delta island in South Korea. We conducted whole genome sequencing of the ES strains and comparative genome analysis on the core clusters of WLP with Acetobacterium woodii DSM1030T and Eubacterium limosum ATCC8486T. The methyl-branch cluster included a formate transporter and duplicates or triplicates copies of the fhs gene, which encodes formyl-tetrahydrofolate synthetase. The formate dehydrogenase cluster did not include the hydrogenase gene, which might be replaced by a functional complex with a separate electron bifurcating hydrogenase (HytABCDE). Additionally, duplicated copies of the acsB gene, encoding acetyl-CoA synthase, are located within or close to the carbonyl-branch cluster. The serum bottle culture showed that ES strains can utilize a diverse range of C1 compounds, including CO, formate, and methanol, as well as CO2. Notably, ES2 exhibited remarkable resistance to high concentrations of C1 substrates, such as 100% CO (200 kPa), 700 mM formate, and 500 mM methanol. Moreover, ES2 demonstrated remarkable growth rates under 50% CO (0.45 h-1) and 200 mM formate (0.34 h-1). These growth rates are comparable to or surpassing those previously reported in other acetogenic bacteria. Our study introduces novel acetogenic ES strains and describes their genetic and physiological characteristics, which can be utilized in C1-based biomanufacturing.

Low-moderate dose whole-brain γ-ray irradiation modulates the expressions of glial fibrillary acidic protein and intercellular adhesion molecule-1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mouse model.

The anti-inflammatory efficacy of radiation therapy (RT) with single fractions below 1.0 Gy has been demonstrated in Alzheimer's disease mouse models. As neuroinflammation is also a major pathological feature of Parkinson's disease (PD), RT may also be effective in PD treatment. Therefore, this study aimed to investigate the anti-inflammatory effect of low-moderate dose RT (LMDRT, 0.6 Gy/single dose, for 5 days) exposure in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg, intraperitoneally, for 5 consecutive days)-induced PD mouse model. Importantly, LMDRT reduced the levels of glial fibrillary acidic protein and intercellular adhesion molecule-1 (CD54) in the striatum region, which increased following MPTP administration. LMDRT also modulated inflammatory gene expression patterns in the substantia nigra region of the MPTP-treated mice. However, LMDRT had no direct effects on the severe loss of dopaminergic neurons and impaired motor behavior in the rotarod test. These results indicate that LMDRT has anti-inflammatory effects by modulating neuroinflammatory factors, including glial fibrillary acidic protein and intercellular adhesion molecule-1, but showed no behavioral improvements or neuroprotection in the MPTP-induced mouse model of PD.

What factors influence continuous usage intention of head-mounted display-based virtual reality content?: a cross-sectional survey.

PURPOSE: The aim of this study was to explore the continuous usage intention of head-mounted display-based virtual reality (HMD-based VR) content among college students. The study also sought to understand how this intention is influenced by factors related to personal cognition, social aspects, VR content, and HMD-related elements. METHODS: This descriptive correlational study used a self-report questionnaire to survey 217 students from two universities in Korea who had prior experience with HMD-based VR content. RESULTS: The mean score for continuous usage intention of HMD-based VR content was 2.59±0.57 points (range, 1-5 points). Regarding the average frequency of HMD-based VR content usage, 64.5% of participants reported using it 1 to 2 times, while 91.7% indicated a total HMD-based VR usage period of less than 6 months. Factors such as personal cognition, VR content, social aspects, and HMD-related elements had explanatory power of 35.1%, 10.7%, 4.4%, and 2.5%, respectively, for the continuous usage intention of HMD-based VR content. Additionally, engagement (ß=.45, p<.001), influential others (ß=.37, p<.001), environmental support (ß=-.18, p=.030), and cyber sickness (ß=-.21, p=.001) were identified as having a significant influence. CONCLUSION: When developing HMD-based VR content, strategies to improve users' personal cognition should be included. Additionally, it is necessary to develop strategies that enhance enjoyment and interest in the content, while also facilitating ongoing social support. Furthermore, coping strategies should be devised that take into account cyber sickness, a potential side effect of these devices.

A Pilot Study to Examine the Effects of a Workplace Cyberbullying Cognitive Rehearsal Mobile Learning Program for Head Nurses: A Quasi-Experimental Study.

This study aimed to (1) develop a version of the cognitive rehearsal program that is suitable for cyberbullying and (2) apply the m-learning method to evaluate its effectiveness among head nurses. This study was conducted in July 2021 and comprised 69 South Korean university hospital head nurses. It was evaluated using a nonequivalent control group pretest-posttest and a quasi-experimental design. The program was developed using the Analysis, Design, Development, Implementation, and Evaluation (ADDIE) step process, consisting of 10 scenarios. Variables with proven reliability were used in the program effect measurement. The differences between the experimental and control groups were examined using an independent t-test (perception) or the Mann-Whitney U test (symptom experience, knowledge, and turnover intention). The program positively affected and improved head nurses' knowledge and perception of workplace cyberbullying; however, it elevated their symptom experience and had no immediate impact on turnover intention. The developed program could be applied as a valuable educational strategy in the nursing field. Head nurses act as intermediaries between individuals and the organization. Therefore, they must respond with in-depth knowledge and perceptions of cyberbullying to fulfill their responsibilities of identifying, mediating, and managing cyberbullying among hospital team members.

Identification and comparison of pandemic-to-symptom networks of South Korea and the United States.

Background: The Coronavirus (COVID-19) pandemic resulted in a dramatic increase in the prevalence of anxiety and depression globally. Although the impact on the mental health of young adults was especially strong, its underlying mechanisms remain elusive. Materials and methods: Using a network approach, the present study investigated the putative pathways between pandemic-related factors and anxiety and depressive symptoms among young adults in South Korea and the U.S. Network analyses were conducted on cross-country data collected during the COVID-19 lockdown period (n = 1,036). Our model included depression symptoms (PHQ-9), generalized anxiety symptoms (GAD-7), and COVID-19-related factors (e.g., COVID-19-related traumatic stress, pandemic concerns, access to medical/mental health services). Results: The overall structure of pandemic-to-symptom networks of South Korea and the U.S. were found to be similar. In both countries, COVID-related stress and negative future anticipation (an anxiety symptom) were identified as bridging nodes between pandemic-related factors and psychological distress. In addition, worry-related symptoms (e.g., excessive worry, uncontrollable worry) were identified as key contributors in maintaining the overall pandemic-to-symptom network in both countries. Conclusion: The similar network structures and patterns observed in both countries imply that there may exist a stable relationship between the pandemic and internalizing symptoms above and beyond the sociocultural differences. The current findings provide new insights into the common potential pathway between the pandemic and internalizing symptoms in South Korea and in the U.S. and inform policymakers and mental health professionals of potential intervention targets to alleviate internalizing symptoms.

Pathogenesis of Alkali Injury-Induced Limbal Stem Cell Deficiency: A Literature Survey of Animal Models.

Limbal stem cell deficiency (LSCD) is a debilitating ocular surface disease that eventuates from a depleted or dysfunctional limbal epithelial stem cell (LESC) pool, resulting in corneal epithelial failure and blindness. The leading cause of LSCD is a chemical burn, with alkali substances being the most common inciting agents. Characteristic features of alkali-induced LSCD include corneal conjunctivalization, inflammation, neovascularization and fibrosis. Over the past decades, animal models of corneal alkali burn and alkali-induced LSCD have been instrumental in improving our understanding of the pathophysiological mechanisms responsible for disease development. Through these paradigms, important insights have been gained with regards to signaling pathways that drive inflammation, neovascularization and fibrosis, including NF-κB, ERK, p38 MAPK, JNK, STAT3, PI3K/AKT, mTOR and WNT/ß-catenin cascades. Nonetheless, the molecular and cellular events that underpin re-epithelialization and those that govern long-term epithelial behavior are poorly understood. This review provides an overview of the current mechanistic insights into the pathophysiology of alkali-induced LSCD. Moreover, we highlight limitations regarding existing animal models and knowledge gaps which, if addressed, would facilitate development of more efficacious therapeutic strategies for patients with alkali-induced LSCD.

Development and Evaluation of a Workplace Bullying Cognitive Rehearsal-Based Nursing Simulation Education Program: A Mixed-Methods Study.

Bullying makes learning difficult for nursing students, and using real-life scenarios during training can improve the understanding of workplace bullying. Thus, to mitigate bullying experienced by nurses, this study developed and evaluated a cognitive rehearsal education program that constituted training nursing students through role-play simulations. A mixed-method research design was used to evaluate 39 nursing students from two universities. A quasi-experimental research design was applied to assess symptoms, knowledge, and perceptions of bullying, and a focus group interview was conducted with six participants. Quantitative analyses revealed that the program improved participants' knowledge and perceptions but not their symptoms. The focus group interview revealed that the program improved participants' coping skills and desire for education. This program could be effective in raising awareness of workplace bullying and increasing relevant coping skills. It can be further developed as part of an overall strategy to manage workplace bullying and its consequences in hospital settings.

Determination and Analysis of Hyper-Variable A Mating Types in Wild Strains of Lentinula edodes in Korea.

The diversity of A mating type in wild strains of Lentinula edodes was extensively analyzed to characterize and utilize them for developing new cultivars. One hundred twenty-three A mating type alleles, including 67 newly discovered alleles, were identified from 106 wild strains collected for the past four decades in Korea. Based on previous studies and current findings, a total of 130 A mating type alleles have been found, 124 of which were discovered from wild strains, indicating the hyper-variability of A mating type alleles of L. edodes. About half of the A mating type alleles in wild strains were found in more than two strains, whereas the other half of the alleles were found in only one strain. About 90% of A mating type combinations in dikaryotic wild strains showed a single occurrence. Geographically, diverse A mating type alleles were intensively located in the central region of the Korean peninsula, whereas only allele A17 was observed throughout Korea. We also found the conservation of the TCCCAC motif in addition to the previously reported motifs, including ATTGT, ACAAT, and GCGGAG, in the intergenic regions of A mating loci. Sequence comparison among some alleles indicated that accumulated mutation and recombination would contribute to the diversification of A mating type alleles in L. edodes. Our data support the rapid evolution of A mating locus in L. edodes, and would help to understand the characteristics of A mating loci of wild strains in Korea and help to utilize them for developing new cultivars.

Description of Fervidibacillus gen. nov. with Two Species, Fervidibacillus albus sp. nov., and Fervidibacillus halotolerans sp. nov., Isolated from Tidal Flat Sediments and Emendation of Misclassificed Taxa in the Genus Caldibacillus.

Two Gram-stain-positive, motile, endospore-forming, facultatively anaerobic strains, designated MEBiC13591T and MEBiC13594T, were isolated from tidal flat sediment of the Incheon City on the west coast of Korea. Growth of both novel strains was observed at pH 5-9 (optimum, pH 7-7.5), and in 0-8% NaCl (optimum, 2% for MEBiC13591T and 3% for MEBiC13594T). Strains MEBiC13591T and MEBiC13594T grew optimally at 50 °C, (37.5-56.1 °C) and 44 °C (20.7-50.7 °C), respectively. The main cellular fatty acids of strain MEBiC13591T were iso-C15: 0, anteiso-C15: 0, iso-C16: 0, iso-C17: 0 and anteiso-C17: 0, while those for strain MEBiC13594T were C14: 0, iso-C14: 0, iso-C15: 0, anteiso-C15: 0 and C16: 0. In both taxa, the major isoprenoid was MK-7. The genomic DNA G + C contents were 34.1 and 37.0 mol% for MEBiC13591T and MEBiC13594T, respectively. A 16S rRNA gene sequence analysis revealed that the novel strains showed high similarity with members of the genera Aeribacillus (95.0%) and Caldibacillus (93.5-94.5%); however, showed lower than 90% with Caldibacillus debilis TfT. Phylogenetic and Phylogenomic analysis revealed that two novel strains comprised distinct phyletic line with members formerly assigned to Caldibacillus. Based on genomic indices, such as AAI and ANI, members formerly affiliated with Caldibacillus and Bacillus as well as the novel strains should be classified into five independent genera. Based on the phenotypic, genomic and biochemical data, strains MEBiC13591T and MEBiC13594T represent two novel species in the novel genus, for which the names Fervidibacillus albus gen. nov., sp. nov. (MEBiC13591T [= KCCM 43317T = KCTC 43181T = JCM 33662T = MCCC 1K04565T]), and Fervidibacillus halotolerans sp. nov. (MEBiC13594T [= KCTC 43182T = JCM 34001T]) are proposed. Three additional genera Caldifermentibacillus, Palidibacillus, and Perspicuibacillus are also proposed by reclassification of the several species with valid names that formerly affiliated with the genera Caldibacillus.

Type 2 diabetes influences intraepithelial corneal nerve parameters and corneal stromal-epithelial nerve penetration sites.

INTRODUCTION: The quantification of intraepithelial corneal basal nerve parameters by in vivo confocal microscopy represents a promising modality to identify the earliest manifestations of diabetic peripheral neuropathy. However, its diagnostic accuracy is hampered by its dependence on neuron length, with minimal consideration for other parameters, including the origin of these nerves, the corneal stromal-epithelial nerve penetration sites. This study sought to utilize high-resolution images of murine corneal nerves to analyze comprehensively the morphological changes associated with type 2 diabetes progression. MATERIALS AND METHODS: ßIII-Tubulin immunostained corneas from prediabetic and type 2 diabetic mice and their respective controls were imaged by scanning confocal microscopy and analyzed automatically for nerve parameters. Additionally, the number and distribution of penetration sites was manually ascertained and the average length of the axons exiting them was computed. RESULTS: The earliest detectable changes included a significant increase in nerve density (6.06 ± 0.41% vs 8.98 ± 1.99%, P = 0.03) and branching (2867.8 ± 271.3/mm2 vs 4912.1 ± 1475.3/mm2 , P = 0.03), and in the number of penetration sites (258.80 ± 20.87 vs 422.60 ± 63.76, P = 0.0002) at 8 weeks of age. At 16 weeks, corneal innervation decreased, most notably in the periphery. The number of penetration sites remained significantly elevated relative to controls throughout the monitoring period. Similarly, prediabetic mice exhibited an increased number of penetration sites (242.2 ± 13.55 vs 305.6 ± 30.96, P = 0.003) without significant changes to the nerves. CONCLUSIONS: Our data suggest that diabetic peripheral neuropathy may be preceded by a phase of neuron growth rather than regression, and that the peripheral cornea is more sensitive than the center for detecting changes in innervation.

Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing new small molecule drugs.

Cell identity changes in ocular surface Epithelia.

Corneal and conjunctival epithelia arise from a common ancestral ectoderm cell, then diverge into distinct lineages. The former develops into a multilayered stratified squamous epithelium, the latter into an expansive mucous membrane that stretches the eyelid margin to the cornea's outskirts. The limbus, which intersects these epithelia, is purported to harbor corneal stem cells. Intrinsic programs that prevent these neighbouring epithelia from mixing and changing identity have not been elucidated, however microenvironmental cues that emanate following tissue damage and ensuing disease, dictate cell fate choices including those that influence form and function. Plasticity of ocular surface epithelia is gauged by their ability to undergo epithelial-mesenchymal transition, transdifferentiation, dedifferentiation and metaplastic transformation. Elucidating the molecular mechanism by which these rare and unusual phenomena arise, and persuading cells to either revert to their original state or remain newly committed, could be exploited into game-changing therapeutics for patients with corneal blindness and other diseases.

BR101801 enhances the radiosensitivity of p53-deficient colorectal cancer cells by inducing G2/M arrest, apoptosis, and senescence in a p53-independent manner.

Inhibition of DNA-dependent protein kinase (DNA-PK) in the non-homologous end-joining repair pathway reportedly increases the radiation sensitivity of cancer cells. We have recently reported that BR101801, a novel triple inhibitor of PI3K-gamma (γ), delta (δ), and DNA-PK, functions as an efficient sensitizer of radiation-induced DNA damage in various human solid cancer cells and a xenograft mouse model. Given that the p53 tumor suppressor gene plays an important role in radiotherapeutic efficacy, in the current study, we focused on the impact of the p53 status on BR101801-induced radiosensitization using isogenic HCT116 p53+/+ and HCT116 p53-/- human colorectal cancer cell lines. In vitro, HCT116 p53+/+ and HCT116 p53-/- human colorectal cancer cells were pretreated with 1 µM BR101801 for 24 h before exposure to ionizing radiation (IR), followed by assays to analyze colony formation, DNA damage, cell cycle changes, senescence, autophagy, apoptosis, and DNA damage response-related proteins. Xenograft mouse models were constructed to examine the potential synergistic effects of BR101801 (50 mg/kg, orally administered once daily) and fractionated IR (2 Gy × 3 days) on tumor growth inhibition in vivo. BR101801 inhibited cell proliferation and prolonged DNA damage in both HCT116 p53+/+ and HCT116 p53-/- human colorectal cancer cells. Combined treatment with BR101801 and IR robustly induced G2/M phase cell cycle arrest, apoptosis, and cellular senescence in HCT116 p53-/- cells when compared with treatment with IR alone. Furthermore, BR101801 synergistically inhibited tumor growth in the HCT116 p53-/- xenograft mouse model. BR101801 enhanced the radiosensitivity of HCT116 human colorectal cancer cells regardless of their p53 status. Moreover, BR101801 exerted robust synergistic effects on IR-induced cell cycle arrest, apoptosis, and tumor growth inhibition, even in radioresistant HCT116 p53-/- cells. Overall, these findings provide a scientific rationale for combining BR101801 with IR as a new therapeutic strategy to overcome radioresistance induced by p53 deficiency.

De novo genome assembly of the bioluminescent mushroom Omphalotus guepiniiformis reveals an Omphalotus-specific lineage of the luciferase gene block.

Omphalotus guepiniiformis, a bioluminescent mushroom species, is a source of the potentially valuable anticancer chemical. To provide genome information, we de novo assembled the high-quality O. guepiniiformis genome using two Next-Generation sequencing techniques, PacBio and Illumina sequencing. Our draft O. guepiniiformis genome comprises 42.5 Mbp of sequence with only 80 contigs and an N50 sequence length of over 1 Mbp. There were 15,554 predicted coding genes, and 7693 genes were functionally annotated with Gene Ontology terms. We performed a genomic study focusing on mushroom bioluminescent pathway cluster genes by comparing 17 luminescent and 23 non-luminescent Agaricales species belonging to 23 genera. Synteny analysis of genomic regions near the luminescent pathway cluster genes inferred that the Omphalotus lineage was genus-specific. In summary, our de novo assembled O. guepiniiformis genome provides significant biological insights into this organism, including the evolution of the luciferase gene block, and forms the basis for future analyses.

Plasticity of ocular surface epithelia: Using a murine model of limbal stem cell deficiency to delineate metaplasia and transdifferentiation.

Maintaining corneal health and transparency are necessary pre-requisites for exquisite vision, a function ascribed to stem cells (SCs) nestled within the limbus. Perturbations to this site or depletion of its SCs results in limbal SC deficiency. While characterizing a murine model of this disease, we discovered unusual transformation phenomena on the corneal surface including goblet cell metaplasia (GCM), conjunctival transdifferentiation, and squamous metaplasia (SQM). GCM arose from K8+ differentiated conjunctival epithelial cells when the limbus was breached and was exacerbated by neovascularization. Regions within the cornea that harbored newly transformed K12+ epithelia were void of blood vessels and GCs, suggesting that the cornea also initiated a self-repair program. Knowledge of the intrinsic circuits that contribute to cell identity change in lineage-restricted epithelia will be invaluable for designing new therapeutics for patients with blinding corneal disease.

Establishment of Genome Based Criteria for Classification of the Family Desulfovibrionaceae and Proposal of Two Novel Genera, Alkalidesulfovibrio gen. nov. and Salidesulfovibrio gen. nov.

Bacteria in the Desulfovibrionaceae family, which contribute to S element turnover as sulfate-reducing bacteria (SRB) and disproportionation of partially oxidized sulfoxy anions, have been extensively investigated since the importance of the sulfur cycle emerged. Novel species belonging to this taxon are frequently reported, because they exist in various environments and are easy to culture using established methods. Due to the rapid expansion of the taxon, correction and reclassification have been conducted. The development of high-throughput sequencing facilitated rapid expansion of genome sequence database. Genome-based criteria, based on these databases, proved to be potential classification standard by overcoming the limitations of 16S rRNA-based phylogeny. Although standards methods for taxogenomics are being established, the addition of a novel genus requires extensive calculations with taxa, including many species, such as Desulfovibrionaceae. Thus, the genome-based criteria for classification of Desulfovibrionaceae were established and validated in this study. The average amino-acid identity (AAI) cut-off value, 63.43 ± 0.01, was calculated to be an appropriate criterion for genus delineation of the family Desulfovibrionaceae. By applying the AAI cut-off value, 88 genomes of the Desulfovibrionaceae were divided into 27 genera, which follows the core gene phylogeny results. In this process, two novel genera (Alkalidesulfovibrio and Salidesulfovibrio) and one former invalid genus ("Psychrodesulfovibrio") were officially proposed. Further, by applying the 95-96% average nucleotide identity (ANI) standard and the 70% digital DNA-DNA hybridization standard values for species delineation of strains that were classified as the same species, five strains have the potential to be newly classified. After verifying that the classification was appropriately performed through relative synonymous codon usage analysis, common characteristics were listed by group. In addition, by detecting metal resistance related genes via in silico analysis, it was confirmed that most strains display metal tolerance.

First report of fusarium wilt disease caused by Fusarium equiseti on grafted watermelon in Korea.

In Korea, most of the grafted watermelons are a fusion of bottle gourd (Lagenaria siceraria) as a rootstock and watermelon (Citrullus lanatus) as a scionstock (Lee et al., 2010). Currently, we have collected several samples from grafted watermelon displaying symptoms of yellowing, withered and wilting leaves. When the symptomatic stem was excised, browning vascular tissues were observed due to the colonization of fungal pathogen. From the samples obtained, 25 fungal isolates were identified as species of Fusarium. Among 25 isolates, 18 were identified as Fusarium oxysporum, four as Fusarium solani, and three as Fusarium equiseti (F. equiseti) . Initial assessment showed that one of the F. equiseti isolates (NIHHS 16-126) was highly virulent to rootstock. Interestingly, this is the first time F. equiseti has been identified pathogenic to grafted watermelon. NIHHS 16-126 isolate was collected from watermelon cultivation field around Buyeo-gun (36.25951°N, 126.92044°E) county. Disease incident was estimated to infect approximately 10% of the watermelon plants cultivated in this area. NIHHS 16-126 isolate was examined to confirm its identity. On potato dextrose agar, colonies appeared yellowish-brown while the aerial mycelium was whitish to peach in color. Macroconidia were relatively long (20.21 - 51.13 × 2.30 - 4.5 µm, n=50), comprise of 3-6 septa, curvature shape and its conidiophores were with monophialides. However, microconidia formation was not observed. These morphological characteristics resemble F. equiseti characters as described by Hyun (2019). For molecular identification, an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (ß-tub) genes were sequenced using primer pairs of ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson 1995), and Bt2a/Bt2b (Carbone and Kohn 1999). BLASTN analysis revealed that ITS-rDNA (LC648248), EF-1α (LC648250), and ß-tub (LC648249) sequences were 99-100% identical to F. equiseti reference sequences (KF515650, KF747331, and KF747330) infected Avicennia marina in China (Lu 2014). Phylogenetic analysis of concatenated ITS-rDNA, EF-1α and ß-tub sequences showed that this isolate clustered in the same clade as F. equiseti, confirming its identity as F. equiseti. For the inoculation, roots of 12-days-old seedlings (watermelon and bottle gourd, n=10 each) were dipped in the conidia suspension (1x106 conidia/µL) for 30 min. Inoculated seedlings were planted in the soil before being transferred to the greenhouse (temperature; 30°C, daylight; 14 hours). Control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated bottle gourd seedlings (n=10) wilted and eventually died. In contrast, none of the inoculated watermelons or control seedlings were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Bottle gourd is the most preferred rootstock for grafted watermelons among Korean farmers due to its ability to resist Fusarium spp. infection. Therefore, the identification of F. equiseti as a fungal that is pathogenic to rootstock is crucial information to manage fusarium wilt disease among grafted watermelon. To our knowledge, this is the first report confirming F. equiseti infection in grafted watermelon plants in Korea.