ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

Cathelicidin ΔPb-CATH4 derived from Python bivittatus accelerates the healing of Staphylococcus aureus-infected wounds in mice.

The effects of ΔPb-CATH4, a cathelicidin derived from Python bivittatus, were evaluated against Staphylococcus aureus-infected wounds in mice. These effects were comparable to those of classical antibiotics. ΔPb-CATH4 was resistant to bacterial protease but not to porcine trypsin. A reduction in the level of inflammatory cytokines and an increase in the migration of immune cells was observed in vitro. Thus, ΔPb-CATH4 can promote wound healing by controlling infections including those caused by multidrug-resistant bacteria via its immunomodulatory effects.

Cometabolic Vinyl Chloride Degradation at Acidic pH Catalyzed by Acidophilic Methanotrophs Isolated from Alpine Peat Bogs.

Remediation of toxic chlorinated ethenes via microbial reductive dechlorination can lead to ethene formation; however, the process stalls in acidic groundwater, leading to the accumulation of carcinogenic vinyl chloride (VC). This study explored the feasibility of cometabolic VC degradation by moderately acidophilic methanotrophs. Two novel isolates, Methylomonas sp. strain JS1 and Methylocystis sp. strain MJC1, were obtained from distinct alpine peat bogs located in South Korea. Both isolates cometabolized VC with CH4 as the primary substrate under oxic conditions at pH at or below 5.5. VC cometabolism in axenic cultures occurred in the presence (10 µM) or absence (<0.01 µM) of copper, suggesting that VC removal had little dependence on copper availability, which regulates expression and activity of soluble and particulate methane monooxygenases in methanotrophs. The model neutrophilic methanotroph Methylosinus trichosporium strain OB3b also grew and cometabolized VC at pH 5.0 regardless of copper availability. Bioaugmentation of acidic peat soil slurries with methanotroph isolates demonstrated enhanced VC degradation and VC consumption below the maximum concentration level of 2 µg L-1. Community profiling of the microcosms suggested species-specific differences, indicating that robust bioaugmentation with methanotroph cultures requires further research.

High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide.

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

Beneficial impacts of residential greenness on sleep deprivation in adults aged 19 or older living in South Korea: A nationwide community health survey in 2011-2018.

BACKGROUND: Evidence of the relationship between greenness and sleep is limited, and, given the worsening sleep insufficiency worldwide, this relationship needs elucidation. In this study, we investigated the association of greenness with sleep deprivation using nationwide survey data. METHODS: This study included 1,727,273 participants in the Korea Community Health Survey who resided in all 229 districts of South Korea from 2011 to 2018. Sleep deprivation variables were defined as strong deprivation or mild deprivation, based on average daily sleep duration of <5 or 5-6 h, respectively. District-specific annual average of satellite-derived enhanced vegetation index (EVI) was used as a green space exposure. A logistic regression with complex survey weights was used to estimate the association between greenness and sleep deprivation, and it was further examined by sex, age group, educational status, income level, and population density. The regression analysis was performed annually, and the annual estimates were pooled by a combined data analysis. RESULTS: A higher level of greenness was associated (odds ratio [95 % confidence interval]) with strong and mild sleep deprivation (0.96 [0.93-0.99] and 0.96 [0.95-0.97]), respectively, and males and the younger age group (<65 years) showed a more prominent association with greenness than in females and the elderly group (65 years or older). In addition, only high-population-density areas showed evident associations of greenness with both strong and mild sleep deprivation. CONCLUSIONS: This large population-based study provides important epidemiological evidence for improving sleep quantity through an increase in greenness exposure and supports policymakers in establishing strategies for urban planning.

Genomewide Analysis and Biological Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity from Three Bat Species.

Cathelicidins are potent antimicrobial peptides with broad spectrum antimicrobial activity in many vertebrates and an important component of the innate immune system. However, our understanding of the genetic variations and biological characteristics of bat cathelicidins is limited. In this study, we performed genome-level analysis of the antimicrobial peptide cathelicidins from seven bat species in the six families, listed 19 cathelicidin-like sequences, and showed that the number of functional cathelicidin genes differed among bat species. Based on the identified biochemical characteristics of bat cathelicidins, three cathelicidins, HA-CATH (from Hipposideros armiger), ML-CATH (from Myotis lucifugus), and PD-CATH (from Phyllostomus discolor), with clear antimicrobial signatures were chemically synthesized and evaluated antimicrobial activity. HA-CATH showed narrow-spectrum antibacterial activity against a panel of 12 reference bacteria, comprising 6 Gram-negative and 6 Gram-positive strains. However, ML-CATH and PD-CATH showed potent antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria with minimum inhibitory concentration (MIC) of 1 and 3 µg/mL, respectively, against Staphylococcus aureus. ML-CATH and PD-CATH also showed antifungal activities against Candida albicans and Cryptococcus cuniculi with MIC of 5 to 40 µg/mL, respectively, and 80% inhibition of the metabolism of Mucor hiemalis hyphae at 80 µg/mL, while displaying minimal cytotoxicity to HaCaT cells. Taken together, although the spectrum and efficacy of bat cathelicidins were species-dependent, the antimicrobial activity of ML-CATH and PD-CATH was comparable to that of other highly active cathelicidins in vertebrates while having negligible cytotoxicity to mammalian cells. ML-CATH and PD-CATH can be exploited as promising candidates for the development of antimicrobial therapeutics.

Development of an Immortalized Porcine Fibroblast Cell Panel With Different Swine Leukocyte Antigen Genotypes.

Immortalized cell lines are valuable resources to expand the molecular characterization of major histocompatibility complex genes and their presented antigens. We generated a panel of immortalized cell lines by transfecting human telomerase reverse transcriptase (hTERT) into primary fibroblast cells prepared from ear, fetal, and lung tissues of 10 pigs from five breeds and successfully cultured them for 30-45 passages. The cell growth characteristic of the immortalized fibroblasts was similar to that of primary fibroblast, which was unable to form colonies on soft agar. The genotypes of major swine leukocyte antigen (SLA) genes, including three classical class I (SLA-1, -2, and -3) and three class II genes (DQB1, DRB1, and DQA), were determined using high-resolution typing. A total of 58 alleles, including a novel allele for SLA-2, were identified. Each cell line was unique. A cell line derived from a National Institutes of Health miniature pig was homozygous across the six major SLA genes. The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on semiquantitative reverse transcription polymerase chain reaction. The immortalized porcine fibroblast cell lines with diverse SLA haplotypes that were developed in this study have potential to be applied in studies regarding the molecular characteristics and genetic structure of SLA genes and epitope-major histocompatibility complex interactions in pigs.

Effects of urbanization on vulnerability to heat-related mortality in urban and rural areas in South Korea: a nationwide district-level time-series study.

BACKGROUND: Although urbanization is often an important topic in climate change studies, the complex effect of urbanization on heat vulnerability in urban and rural areas has rarely been studied. We investigated the disparate effects of urbanization on heat vulnerability in urban and rural areas, using nationwide data. METHODS: We collected daily weather data for all 229 administrative districts in South Korea (2011-17). Population density was applied as an urbanization indicator. We calculated the heat-mortality risk using a distributed lag nonlinear model and analysed the relationship with population density. We also examined district characteristics that can be related to the spatial heterogeneity in heat-mortality risk. RESULTS: We found a U-shaped association between population density and heat-mortality risk, with the highest risk for rural populations; in urban areas, risk increases with increasing population density. Higher heat-mortality risk was associated with a lower number of hospital beds per person and higher percentage of people requiring recuperation. The association between hospital beds and heat-mortality risk was prominent in high-density urban areas, whereas the association between the percentage of people requiring recuperation and heat-mortality risk was pronounced in rural areas. CONCLUSIONS: Our findings indicate that the association between population density and heat-mortality risk is different in urban and rural areas, and that district characteristics related to heat-mortality risk also differ by urbanicity. These results can contribute to understanding the complex role of urbanization on heat vulnerability and can provide evidence to policy makers for prioritizing resources.

Comparison of DNA/RNA yield and integrity between PMAP36-mediated and other bacterial lysis methods.

Currently, several methods are available for the isolation of bacterial DNA and RNA. However, the diversity and complexity of cell envelope structures limit their efficiency depending on the target bacterial species. In this study, we compared the differences in yield and integrity of RNA prepared from four gram-negative and six gram-positive bacterial species using bead-beating, bacteriolytic protein, and PMAP36-vortexing methods. Similarly, we also compared the efficiency of DNA extraction from Staphylococcus aureus. Physical disruption of bacterial cells showed versatility in breaking cells against all tested species; however, a decrease in the integrity of isolated DNA and RNA was observed. Among membranolytic proteins, PMAP36 showed the most promising results, in terms of both the yield and integrity of the prepared nucleic acids. Our results show that each method has inherent advantages and disadvantages depending on its application. Therefore, the characteristics of each method and target species should be considered before the extraction of bacterial DNA and RNA.

Roles of urban heat anomaly and land-use/land-cover on the heat-related mortality in the national capital region of South Korea: A multi-districts time-series study.

The urban heat anomaly has been suggested as a representative risk factor for human health in metropolitan areas, but few studies have measured a quantitative increase in risk due to the urban heat anomaly on heat-related mortality in the summer season or assessed the role of various types of land-use/land-cover (LULC), which may contribute to the urban heat anomaly. In this study, we evaluated the association between the urban heat anomaly and heat-related mortality risk in the summer and the potential roles of multiple types of LULC indicators. We used district-level time-series and cadastral data from 51 urban districts in the national capital region of South Korea. We applied a two-stage analysis. In the first stage, we estimated the district-specific heat-related mortality risk by using a distributed lag non-linear model. In the second stage, we used a meta-analysis to pool the estimates across all districts and calculate the association between the urban heat anomaly/LULC indicators and heat-related mortality risk. We found that the higher urban heat anomaly was related to lower vegetation and higher urban surface indicators, and the urban heat anomaly was positively associated with the heat-related mortality risk. The association between the urban heat anomaly and the heat-related mortality risk was more pronounced in the elderly (age ≥ 65 years) and female population than in the non-elderly and male population. We also found that the LULC indicators affected the heat-related mortality only through the urban heat anomaly. Our findings indicate that urban areas may be more vulnerable to heat-related mortality risk as determined by the urban heat anomaly. These results suggest a need for urban heat mitigation strategies such as increased vegetation or surface albedo to help reduce heat-related mortality risk.

Heatwave-Related Mortality Risk and the Risk-Based Definition of Heat Wave in South Korea: A Nationwide Time-Series Study for 2011-2017.

Studies on the pattern of heatwave mortality using nationwide data that include rural areas are limited. This study aimed to assess the risk of heatwave-related mortality and evaluate the health risk-based definition of heatwave. We collected data on daily temperature and mortality from 229 districts in South Korea in 2011-2017. District-specific heatwave-related mortality risks were calculated using a distributed lag model. The estimates were pooled in the total areas and for each urban and rural area using meta-regression. In the total areas, the threshold point of heatwave mortality risk was estimated at the 93rd percentile of temperature, and it was lower in urban areas than in rural areas (92nd percentile vs. 95th percentile). The maximum risk of heatwave-related mortality in the total area was 1.11 (95% CI: 1.01-1.22), and it was slightly greater in rural areas than in the urban areas (RR: 1.23, 95% CI: 0.99-1.53 vs. RR: 1.10, 95% CI: 1.01-1.20). The results differ by age- and cause-specific deaths. In conclusion, the patterns of heatwave-related mortality risk vary by area and sub-population in Korea. Thus, more target-specific heatwave definitions and action plans should be established according to different areas and populations.

Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells.

The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate-buffered saline (PBS) was employed, with a slow-ramp freezing rate. We observed > 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive-cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze-thawing process. Real-time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis- and transcription-related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long-term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright © 2014 John Wiley & Sons, Ltd.

Successful Localization of Intraoral Foreign Body with C-arm Fluoroscopy.

During surgical procedures, unexpected material, including surgical instruments and tissue segments, may get lost in the surgical field. Most of these should be immediately removed to prevent further complications, such as vital organ irritation, infection, and inflammatory pseudo-tumor formation. However, it is not always easy to define the exact location of the foreign body, especially if the item is very small and/or it is embedded in the soft tissue of the head and neck region. Intraoperative real-time radiological imaging with C-arm fluoroscopy can be useful to trace the three-dimensional location of small and embedded foreign bodies in the oral and maxillofacial area. We describe an unusual case of an embedded micro-screw in the intrinsic tongue muscle that had been dropped into the sublingual space during a lower alveolar bone graft procedure. The lost foreign body was accurately identified with C-arm fluoroscopy and safely removed without any further complications.

Co-development of pyogenic granuloma and capillary hemangioma on the alveolar ridge associated with a dental implant: a case report.

INTRODUCTION: The development of various benign oral mucosal lesions associated with dental implants, such as pyogenic granuloma or peripheral giant cell granuloma, has been rarely reported. However, the occurrence of vascular diseases, such as hemangioma, related to dental implants has not been explored in the literature. In this study, we report a case of co-development of pyogenic granuloma and capillary hemangioma on the alveolar ridge associated with a dental implant in a patient undergoing antithrombotic therapy. To the best of our knowledge, this is first case of hemangioma formation associated with a dental implant. CASE PRESENTATION: A 68-year-old Korean man was referred for intermittent bleeding and a dome-shaped overgrowing mass on his upper alveolar ridge. He underwent dental implantation 5 years ago, and was started on warfarin for cerebral infarction a year ago. He had experienced gum bleeding and gingival mass formation 6 months after warfarinization; then, his implant fixture was removed. However, his gingival mass has been gradually increasing. The gingival mass was surgically excised, and revealed the coexistence of pyogenic granuloma and capillary hemangioma in histological analysis of the specimen. The lesion has showed no recurrence for more than a year. CONCLUSIONS: Regarding immunostaining features, the endothelial cell markers, CD34 and CD31, and the mesenchymal cell marker, vimentin, were strongly detected, but cell proliferation marker, Ki-67, was negatively expressed in the endothelial cells of the hemangioma portion. However, in the pyogenic granuloma portion, CD34 was almost negatively detected, whereas vimentin and Ki-67 were highly detected in the fibroblast-like tumor cells. According to these heterogeneous characteristics of the lesion, the patient was diagnosed with coexistence of pyogenic granuloma and capillary hemangioma associated with the dental implant on the attached gingiva. We recommend that patients with dental implants who have chronic peri-implantitis under antithrombotic therapy should be closely followed to ensure early detection of oral mucosal abnormalities.

Expansile keratocystic odontogenic tumor in the maxilla: immunohistochemical studies and review of literature.

Keratocystic odontogenic tumors (KCOT) - previously termed odontogenic keratocysts (OKC) - are characterized by aggressive behavior and a high rate of recurrence. Histopathologically, the basal layer of KCOT shows a higher cell proliferation rate and increased expression of anti-apoptosis genes. Clinically, KCOT is frequently involved in the mandibular posterior region but is not common in the posterior maxilla. However, it should be noted that due to its expansive characteristics, KCOT involved near the maxillary sinus could easily expand to an enormous size and occupy the entire maxilla. To achieve total excision of these expanded cystic tumors in the maxilla, a more aggressive approach would be needed. In this report, we describe two cases of expansile KCOT involving the entire unilateral maxilla and maxillary sinus; they were completely excised using the Weber-Ferguson approach, showing no evidence of recurrence during the follow-up period of more than two years. In immunohistochemical analyses of the tumor specimens, p53 and p63 showed strong expression, and B-cell lymphoma 2 (BCL2) and MKI67 (Ki-67) showed moderate or weak expression, however, detection of BCL2-associated X protein (BAX) was almost negative. These data indicate that expansile KCOT possesses increased anti-apoptotic activity and cell proliferation rate but decreased apoptosis. These properties of KCOT may contribute to tumor enlargement, aggressive behavior, and high recurrence rate.