First Report of Passiflora Latent Virus Infecting Passion Fruit (Passiflora edulis) in South Korea.

Passion fruit (Passiflora edulis) viral diseases caused by papaya leaf curl Guangdong virus, cucumber mosaic virus, East Asian Passiflora virus, and euphorbia leaf curl virus have been reported in South Korea (Joa et al. 2018; Kim et al. 2018). In June 2021, virus-like symptoms, e.g., mosaic pattern, curling, chlorosis, and deformation, were observed on leaves and fruits of greenhouse-grown P. edulis in Iksan, South Korea, with disease incidence greater than 2% (300 plants: 8 symptomatic plants and 292 asymptomatic plants). Total RNA was extracted from a pooled sample of symptomatic leaves of an individual P. edulis plant using the RNeasy Plant Mini Kit (Qiagen, Germany), and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA). Next-Generation Sequencing (NGS) was performed using the Illumina NovaSeq 6000 system (Macrogen Inc., Korea). De novo assembly of the resulting 121,154,740 reads was performed using Trinity (Grabherr et al. 2011). A total of 70,895 contigs was assembled (>200 bp) and annotated against the NCBI viral genome database using BLASTn (ver. 2.12.0). One 827-nt contig was annotated as milk vetch dwarf virus (MVDV), a member of the genus Nanovirus in the family Nanoviridae (Bangladesh isolate, acc. no. LC094159, 96.0% nucleotide identity), and the other 3,639-nt contig corresponded to Passiflora latent virus (PLV), a member of the genus Carlavirus in the family Betaflexiviridae (Israel isolate, acc. no. DQ455582, 90.0% nucleotide identity). For further confirmation, total RNA was isolated from symptomatic leaves of the same P. edulis used for NGS analysis using a viral gene spin DNA/RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea), and reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers: PLV-F/R (5'-GTGCCCACCGAACATGTTACCTC-3'/5'-CCATGCACTTGGAATGCTTACCC-3') targeting the coat protein region of PLV, MVDV-M-F/R (5'-CTAGTCAGCCATCCAATGGTG-3'/5'-GTGCAGGGTTTGATTGTCTGC-3') targeting the movement protein region, and MVDV-S-F/R (5'-GGATTTTAATACGCGTGGACGATC-3'/5'-AACGGCTATAAGTCACTCCGTAC-3') targeting the coat protein region of MVDV. An expected PCR product of 518 bp corresponding to PLV was amplified, while MVDV was not detected. The amplicon was directly sequenced, and its nucleotide sequence was deposited in GenBank (acc. no. OK274270). A BLASTn analysis showed that the nucleotide sequence of the PCR product shared 93.0% and 96.2% identity with PLV isolates from Israel (MH379331) and Germany (MT723990), respectively. In addition, six passion fruit leaves and two fruit samples with PLV-like symptoms were collected from a total of eight plants grown in the greenhouse in Iksan for RT-PCR analysis, and six samples tested positive for PLV. However, PLV was not detected in one leaf and one fruit among all samples. Mechanical sap inoculation was conducted using extracts of systemic leaves as inoculum on P. edulis and the indicator plants Chenopodium quinoa, Nicotiana benthamiana, N. glutinosa, and N. tabacum. In P. edulis, vein chlorosis and yellowing on systemic leaves were observed 20 days post inoculation (dpi). Necrotic local lesions were observed on inoculated leaves of N. benthamiana and N. glutinosa 15 dpi, and PLV infection was confirmed by RT-PCR assay in symptomatic leaf tissue. This study aimed to determine whether commercially grown passion fruit in the southern part of South Korea could be infected with and potentially spread PLV. Whereas PLV was asymptomatic in persimmon (Diospyros kaki) in South Korea, no pathogenicity testing in passion fruit was reported (Cho et al. 2021). Here, we have shown the natural infection of passion fruit with PLV in South Korea for the first time and associated infection with obvious symptoms. This suggests a need to evaluate potential losses in passion fruit and the selection of healthy propagation material.

ß-Cell protection and antidiabetic activities of Crassocephalum crepidioides (Asteraceae) Benth. S. Moore extract against alloxan-induced oxidative stress via regulation of apoptosis and reactive oxygen species (ROS).

BACKGROUND: Medicinal plants are becoming more popular in the treatment of various diseases because of the adverse effects of the current therapy, especially antioxidant plant components such as phenols and flavonoids have a protective role against oxidative stress-induced degenerative diseases like diabetes. Thus, the purpose of this study was to investigate ß-cell protection and antidiabetic activities of Crassocephalum crepidioides (Asteraceae) Benth. S. Moore. METHOD: The in-vitro study was conducted by the pancreatic ß-cell culture and α-amylase inhibition technique which includes two methods, namely starch-iodine method and 3,5-dinitrosalicylic acid (DNSA) method. On the other hand, the in-vivo study was performed by oral glucose tolerance test (OGTT) method and alloxan-induced diabetes method by using Wistar albino rat. At the end pancreatic specimens were removed and processed for histopathological study. RESULT: The plant extract showed significant (*p < 0.05, **p < 0.01) effect on hyperglycemia as compared to standard (Gliclazide) in OGTT. The plant extract showed efficient protection activity of pancreatic ß-cell from cell death in INS-1 cell line by significantly reduced (*p < 0.05, **p < 0.01) the levels alloxan-induced apoptosis and intracellular reactive oxygen species (ROS) accumulation. In addition, the plant extract showed a significant (*p < 0.05, **p < 0.01) effect on hyperglycemia by increases in percent of ß-cells present in each islet (45% - 60%) compared to the diabetic group. CONCLUSION: The result showed that C. crepidioides had ß-cell protection and antidiabetic activities in pancreatic ß-cell culture and Wistar albino rat.

Polyphenolic Extract of Euphorbia supina Attenuates Manganese-Induced Neurotoxicity by Enhancing Antioxidant Activity through Regulation of ER Stress and ER Stress-Mediated Apoptosis.

Manganese (Mn) is an important trace element present in human body, which acts as an enzyme co-factor or activator in various metabolic reactions. While essential in trace amounts, excess levels of Mn in human brain can produce neurotoxicity, including idiopathic Parkinson's disease (PD)-like extrapyramidal manganism symptoms. This study aimed to investigate the protective role of polyphenolic extract of Euphorbia supina (PPEES) on Mn-induced neurotoxicity and the underlying mechanism in human neuroblastoma SKNMC cells and Sprague-Dawley (SD) male rat brain. PPEES possessed significant amount of total phenolic and flavonoid contents. PPEES also showed significant antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and reducing power capacity (RPC) assays. Our results showed that Mn treatment significantly reduced cell viability and increased lactate dehydrogenase (LDH) level, which was attenuated by PPEES pretreatment at 100 and 200 µg/mL. Additionally, PPEES pretreatment markedly attenuated Mn-induced antioxidant status alteration by resolving the ROS, MDA and GSH levels and SOD and CAT activities. PPEES pretreatment also significantly attenuated Mn-induced mitochondrial membrane potential (ΔΨm) and apoptosis. Meanwhile, PPEES pretreatment significantly reversed the Mn-induced alteration in the GRP78, GADD34, XBP-1, CHOP, Bcl-2, Bax and caspase-3 activities. Furthermore, administration of PPEES (100 and 200 mg/kg) to Mn exposed rats showed improvement of histopathological alteration in comparison to Mn-treated rats. Moreover, administration of PPEES to Mn exposed rats showed significant reduction of 8-OHdG and Bax immunoreactivity. The results suggest that PPEES treatment reduces Mn-induced oxidative stress and neuronal cell loss in SKNMC cells and in the rat brain. Therefore, PPEES may be considered as potential treat-ment in Mn-intoxicated patients.

Turmeric extract and its active compound, curcumin, protect against chronic CCl4-induced liver damage by enhancing antioxidation.

BACKGROUND: Curcumin, a major active component of turmeric, has previously been reported to alleviate liver damage. Here, we investigated the mechanism by which turmeric and curcumin protect the liver against carbon tetrachloride (CCl4)-induced injury in rats. We hypothesized that turmeric extract and curcumin protect the liver from CCl4-induced liver injury by reducing oxidative stress, inhibiting lipid peroxidation, and increasing glutathione peroxidase activation. METHODS: Chronic hepatic stress was induced by a single intraperitoneal injection of CCl4 (0.1 ml/kg body weight) into rats. Turmeric extracts and curcumin were administered once a day for 4 weeks at three dose levels (100, 200, and 300 mg/kg/day). We performed ALT and AST also measured of total lipid, triglyceride, cholesterol levels, and lipid peroxidation. RESULT: We found that turmeric extract and curcumin significantly protect against liver injury by decreasing the activities of serum aspartate aminotransferase and alanine aminotransferase and by improving the hepatic glutathione content, leading to a reduced level of lipid peroxidase. CONCLUSIONS: Our data suggest that turmeric extract and curcumin protect the liver from chronic CCl4-induced injury in rats by suppressing hepatic oxidative stress. Therefore, turmeric extract and curcumin are potential therapeutic antioxidant agents for the treatment of hepatic disease.

Comparison of TGF-ß, PDGF, and CTGF in hepatic fibrosis models using DMN, CCl4, and TAA.

Three chemotoxins including dimethylnitrosamine (DMN), carbon tetrachloride (CCl4), and thioacetamide (TAA) are commonly used in hepatofibrotic models. We aimed to draw characteristics of histopathology and pro-fibrogenic cytokines including TGF-ß, PDGF and CTGF among three models. Rats were divided into six groups and intra-peritoneally injected with DMN (10 mg/kg, for three weeks, three consecutive days weekly), CCl4 (1.6 g/kg, for 10 weeks, twice weekly), TAA (200 mg/kg, for 12 weeks, twice weekly) or their corresponded treatment for each control group. The liver weights were decreased in DMN model, but not other models. Ascites were occurred as 3-, 2-, and 7-rats in DMN, CCl4, and TAA model, respectively. The lipid peroxidation was highest in CCl4 model, serum levels of liver enzymes were increased as similar severity. The hepatofibrotic alterations were remarkable in DMN and TAA model, but not CCl4 as evidenced by the Masson trichrome staining and hydroxyproline. The immunohistochemistry for α-SAM showed that the DMN model was most severely enhanced than other models. On the other hand, hepatic tissue levels of pro-fibrogenic cytokines including TGF-ß, PDGF, and CTGF were generally increased in three models, but totally different among models or measurement resources. Especially, serum levels of three cytokines were remarkably increased by CCl4 injection and CTGF levels in both hepatic tissue and serum were highest in CCl4 group. Our results firstly demonstrated comparative study for features of morphological finding and pro-fibrogenic cytokines in serum and hepatic protein levels among three models. Above results would be a helpful reference for hepatofibrotic studies.

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system.

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the Gateway(TM) system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.

Modulation of radiation-induced alterations in oxidative stress and cytokine expression in lung tissue by Panax ginseng extract.

We investigated the modulating effect of Panax ginseng extract (PGE) on radiation-induced lung injury (RILI) by measuring early changes in oxidative stress levels, cytokine expression, and the histopathology of mouse lung tissue treated with high dose of X-ray radiation. The mice were pretreated with 25, 50, and 100-mg/kg doses of PGE orally for four consecutive days, and their thoraces were then exposed to 15-Gy X-ray radiation 1 h after the last administration of PGE on day 4. The pretreatments with 50 and 100 mg/kg PGE led to significant reductions in the elevation of lipid peroxidation levels at 2 and 10 days, respectively, after irradiation. The mice pretreated with PGE exhibited dose-dependent reductions in the irradiation-induced production of tumor necrosis factor α and transforming growth factor ß1 cytokines 10 days after irradiation, with these reductions nearly reaching the control levels after the 100-mg/kg dose. Furthermore, together with providing significant protection against reductions in catalase activity and glutathione content, pretreatment with 100 mg/kg PGE resulted in a marked attenuation of the severity of inflammatory changes in lung tissue 10 days after irradiation. A high pretreatment dose of PGE may be a useful pharmacological approach for protection against RILI.

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

Bacillus anthracis genomic DNA enhances lethal toxin-induced cytotoxicity through TNF-α production.

BACKGROUND: Bacillus anthracis is the etiological agent of anthrax. Lethal toxin (LT) produced by B. anthracis is a well-known key virulence factor for anthrax because of its strong cytotoxic activity. However, little is known about the role of B. anthracis genomic DNA (BAG) in anthrax pathogenesis. RESULTS: We examined the effect of BAG on TNF-α production and LT-mediated cytotoxicity during B. anthracis spore infection in mouse macrophage cell lines (RAW264.7 cells and J774A.1) and BALB/c mice. Infection of RAW264.7 cells with B. anthracis spores induced TNF-α expression in a multiplicity of infection (MOI)-dependent manner, and this enhancement was attenuated by the toll-like receptor (TLR) 9 inhibitor oligodeoxynucleotide (ODN)2088. BAG led to TNF-α expression in a dose- and time-dependent manner when applied to RAW264.7 cells. TNF-α expression induced by BAG was reduced by either pretreatment with TLR9 inhibitors (ODN2088 and chloroquine (CQ)) or transfection with TLR9 siRNA. Furthermore, BAG-induced TNF-α production in TLR9(+/+) macrophages was completely abrogated in TLR9(-/-) macrophages. BAG enhanced the phosphorylation of mitogen-activated protein kinases (MAPK), and BAG-induced TNF-α expression was attenuated by pretreatment with MAPK inhibitors. A reporter gene assay and confocal microscopy demonstrated that BAG increased NF-κB activation, which is responsible for TNF-α expression. Treatment with BAG alone showed no cytotoxic activity on the macrophage cell line J774A.1, whereas LT-mediated cytotoxicity was enhanced by treatment with BAG or TNF-α. Enhanced LT-induced lethality was also confirmed by BAG administration in mice. Furthermore, LT plus BAG-mediated lethality was significantly recovered by administration of Infliximab, an anti-TNF-α monoclonal antibody. CONCLUSIONS: Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.

Larval chigger mites collected from small mammals in 3 provinces, Korea.

A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun, Gyeonggi-do (Province) (2,754 mites from 30 small mammals), Asan city, Chungcheongnam-do (3,358 mites from 48 mammals), and Jangseong-gun, Jeollanam-do (3,169 for 62 mammals) from April-November 2009 in the Republic of Korea (= Korea) and were identified to species. Leptotrombidium pallidum was the predominant species in Hwaseong (95.8%) and Asan (61.2%), while Leptotrombidium scutellare was the predominant species collected from Jangseong (80.1%). Overall, larval chigger mite indices decreased from April (27.3) to June (4.9), then increased in September (95.2) and to a high level in November (169.3). These data suggest that L. pallidum and L. scutellare are the primary vectors of scrub typhus throughout their range in Korea. While other species of larval chigger mites were also collected with some implications in the transmission of Orientia tsutsugamushi, they only accounted for 11.2% of all larval chigger mites collected from small mammals.

Spotted fever group rickettsia closely related to Rickettsia monacensis isolated from ticks in South Jeolla province, Korea.

Rickettsia monacensis, a spotted fever group rickettsia, was isolated from Ixodes nipponensis ticks collected from live-captured small mammals in South Jeolla province, Korea in 2006. Homogenates of tick tissues were inoculated into L929 and Vero cell monolayers using shell vial assays. After several passages, Giemsa staining revealed rickettsia-like organisms in the inoculated Vero cells, but not the L929 cells. Sequencing analysis revealed that the ompA-small part (25-614 bp region), ompA-large part (2849-4455 bp region), nearly full-length ompB (58-4889 bp region) and gltA (196-1236 bp region) of the isolates had similarities of 100%, 99.8%, 99.3% and 99.5%, respectively, to those of R. monacensis. Furthermore, phylogenetic analysis showed that the isolate was grouped into the cluster in the same way as R. monacensis in the trees of all genes examined. These results strongly suggest that the isolate is closely related to R. monacensis. As far as is known, this is the first report of isolation of R. monacensis from ticks in Korea.

Detection of Rickettsia monacensis from Ixodes nipponensis collected from rodents in Gyeonggi and Gangwon Provinces, Republic of Korea.

A total of 1,305 ticks were collected from wild rodents captured monthly, except July and August, during 2008 at three US-ROK operated military training sites and three US military installations in Gyeonggi and Gangwon Provinces, the Republic of Korea (ROK). Ixodes nipponensis was the most frequently collected tick (n = 1,299, 99.5 %), followed by Ixodes pomerantzevi (n = 6, 0.5 %). The ticks were pooled (1-15/sample) and tested by nested polymerase chain reaction (nPCR) for spotted fever group (SFG) rickettsiae with primer sets targeting the outer membrane protein B (ompB), citrate synthase (gltA), and 17-kDa antigen gene loci. A total of 115/197 (58.4 %) pools were positive by nPCR for the outer membrane protein ompB. Nucleotide sequence analysis of 105/115 (91.3 %) ompB targeted nPCR positive products showed a high degree of similarity to Rickettsia monacensis (99.3-100 %, n = 87) and R. japonica (99.5-100 %, n = 18). From the 87 positive samples demonstrating a high degree of similarity to R. monacensis, 15 were selected and analyzed by nPCR for gltA and the 17-kDa genes. A total of 12/15 pooled samples were positive for by nPCR for gltA, with amplicons demonstrating a high degree of similarity to R. monacensis (99.3-99.7 %). A total of 13/15 pooled samples were positive by nPCR for the 17-kDa gene, with amplicons demonstrating a high degree of similarity to R. monacensis (99.4-100 %). These findings demonstrate that R. monacensis is distributed throughout Gyeonggi and Gangwon Provinces in the ROK. Furthermore, data suggest a relative high prevalence of R. monacensis in the tick, I. nipponensis.

Hepatoprotective effect of Amomum xanthoides against dimethylnitrosamine-induced sub-chronic liver injury in a rat model.

CONTEXT: Amomum xanthioides Wall. ex Baker (Zingiberaceae) is a tropical medicinal plant that is commonly utilized in the treatment of digestive system disorders in Asia for a long time. OBJECTIVE: This study aimed to evaluate the hepatoprotective effect and related mechanisms of A. xanthoides. MATERIALS AND METHODS: Sub-chronic liver injury was induced by dimethylnitrosamine (DMN, 10 mg/kg, three times per week for 3 weeks, i.p.) in rats. Water extract of A. xanthoides (WAX, 50 and 100 mg/kg) was given once a day for 3 weeks. RESULTS AND CONCLUSION: WAX (100 mg/kg) significantly attenuated the DMN-induced excessive release of alanine aminotransferase (123.6 IU/L), aspartate aminotransferase (227.9 IU/L), alkaline phosphatase (820.9 IU/L) and total bilirubin (0.50 g/dL) in serum (p < 0.01), and hydroxyproline (30.5 mg/g tissue) and malondialdehyde (MDA) (53.6 µM/g tissue) contents (p < 0.01) in liver tissue. Furthermore, WAX significantly ameliorated the depletion of total antioxidant capacity (2.54 µM/mg tissue), superoxide dismutase (0.30 U/mg tissue), glutathione (2.10 µM/mg tissue) and catalase (605.0 U/mg tissue) activities (p < 0.05 or p < 0.01) in liver tissue. Histopathological and immunohistochemical analyses indicated that WAX markedly reduced inflammation, necrosis, collagen accumulation and activation of hepatic satellite cells in the liver. Our findings demonstrated that A. xanthoides exerts favorable hepatoprotective effects via positive regulation of the antioxidative system.

Evaluation of the Weeds around Capsicum annuum (CA) Cultivation Fields as Potential Habitats of CA-Infecting Viruses.

Capsicum annuum (CA) is grown outdoors across fields in Jeollabuk-do, South Korea. The weeds surrounding these fields were investigated regarding the infection of 11 viruses infecting CA during the year 2014-2018. In the reverse transcription polymerase chain reaction diagnosis, 546 out of 821 CA samples (66.5%) were infected by nine viruses, and 190 out of 918 weed samples (20.7%) were infected by eight viruses. Correlation analysis of the mutual influence of the viruses infecting CA and weeds during these 5 years showed that five viruses had significant positive correlations with the infection in both CA and weeds. Over the study period, the weeds infected by cucumber mosaic virus (CMV) in the previous year were positively correlated with the incidence of CMV infection in CA in the current year, although the correlation was lower for tomato spotted wilt virus (TSWV) compared to CMV. The CMV infection percent was 14.0% in summer annuals, 11.4% in perennials, and 7.8% in winter annuals. However, considering the overwintering period without CA, the infection percent was 5.2% higher in winter annuals and perennials than that in summer annuals, indicating that winter annual and perennial weeds served as the main habitats for insect vectors. The TSWV infection percent in weeds was 10.4% in summer annuals, 6.4% in winter annuals, and 6.2% in perennials. The weeds surrounding CA fields, acting as the intermediate hosts, were found to be the potent sources of infection, influencing the spread and diversity of CA-infecting viruses. The results of this study can contribute to prevent viral infection in agricultural fields.

Targeted Sequencing of Ascites and Peritoneal Washing Fluid of Patients With Gastrointestinal Cancers and Their Clinical Applications and Limitations.

Cytology from gastrointestinal (GI) cancers is frequently obtained from ascites and peritoneal washing fluids. Examination of ascites and peritoneal washing fluids from patients with GI cancers can help in the tumor staging and prognosis. Tumor-derived DNA in these cytology samples can be a target for next generation sequencing (NGS). Targeted NGS was evaluated in ascites and peritoneal washing samples obtained from 33 patients with GI cancers. These sequences were compared with those from tumor tissue samples, and correlated with cytopathologic findings of the ascites and peritoneal fluid samples. The correlation between fluid and tissue genotyping results was 25%, with a sensitivity of 21.43%. The volume of tumor contained within the fluid samples was low, ranging from ~0 to 10%. Importantly, the sensitivity of detection of somatic mutations in the fluid samples could be increased to 69.2% by assessing samples containing >2% tumor volume. Evaluation of cells from ascitic fluid showed the presence of KRAS, TP53, and CDH1 mutations in 33, 13, and 7%, respectively, of patients with pancreatic cancer, and the presence of KRAS, TP53, and APC mutations in 25, 12, and 13%, respectively, of patients with gastric cancer. Ascites of one of the latter patients acquired KRAS mutation, which was a novel mutation during metastasis. Targeted NGS of ascites and peritoneal washing fluid have clinical implications, as well as limitations, in patients with GI cancers. NGS-based cytology examination may expand cytomolecular practices in GI cancer patients.

Protection and Response of a Tertiary Hospital in South Korea to the COVID-19 Outbreak.

OBJECTIVE: Here, we present an overview of how a tertiary hospital responded to maintain necessary activities and protect patients and staff from the coronavirus disease (COVID-19) outbreak. METHODS: Gil Medical Center, a tertiary hospital in Incheon, has operated a special response team since January 21, 2020. All visitors were assessed for body temperature and respiratory symptoms, and screened for recent overseas travel. Suspected COVID-19 patients were taken to a screening clinic. All febrile patients with or without respiratory symptoms were taken to a respiratory safety clinic. An isolation ward, which consisted of 10 negative-pressure rooms, was used to treat confirmed cases. More than 120 beds were prepared for the outbreak, and patients with pneumonia were preemptively isolated. RESULTS: By May 5, 480 960 visitors were assessed at the control station, 3350 patients visited the triage center, and 1794 were treated in the respiratory safety clinic. Seventeen confirmed cases were admitted to the negative isolation ward, and 350 patients with pneumonia were preemptively isolated. A total of 2977 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction tests were performed. CONCLUSIONS: While tertiary hospitals play an important role in treating both COVID-19 patients and non-COVID-19 patients, hospital staff have to protect themselves from unexpected in-hospital transmission. A multifaceted response must be undertaken to protect tertiary hospitals and their staff during the COVID-19 epidemic.

Molecular mechanisms underlying the effects of the small molecule AMC-04 on apoptosis: Roles of the activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway.

The unfolded protein response (UPR) is an emerging target pathway for cancer treatment owing to its ability to induce cell death. In our previous analysis of UPR-modulating small molecules, we had reported that piperazine oxalate derivative compounds (AMC-01-04) are able to promote increased phosphorylation of eukaryotic translation initiation factor-2 alpha (eIF2α). In this study, we found that AMC-04 induces apoptotic cell death via the activation of UPR in human breast and liver cancer cells. AMC-04 upregulated the expression of activating transcription factor-4 (ATF4)-C/EBP homologous protein (CHOP) and death receptor 5 (DR5) in cancer cells, as revealed by microarray analysis, small-interference RNA assay, and western blotting. From a mechanistic perspective, cytotoxic UPR pathway activation by AMC-04 is mediated by reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (p38 MAPK) signaling. A chemical informatics approach predicted that AMC-04 modulates histone methyltransferase activity. Based on biochemical analysis, the activity of histone methyltransferases, including SUV39H1, SUV39H2, SETDB1, and EHMT1, was inhibited by AMC-04. Furthermore, chemical inhibition of the identified target proteins induced UPR activation and apoptotic cell death, suggesting that inhibition of histone methyltransferases is a promising strategy for cancer therapy. Taken together, we showed that the small molecule AMC-04 modulates epigenetic enzyme activity and mediates the link between cytotoxic UPR and histone modifications.

Identification of a Clinical Cutoff Value for Multiplex KRASG12/G13 Mutation Detection in Colorectal Adenocarcinoma Patients Using Digital Droplet PCR, and Comparison with Sanger Sequencing and PNA Clamping Assay.

KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) is a major predictive marker for anti-epidermal growth factor receptor treatment, and determination of KRAS mutational status is crucial for successful management of colorectal adenocarcinoma. More standardized and accurate methods for testing KRAS mutation, which is vital for therapeutic decision-making, are required. Digital droplet polymerase chain reaction (ddPCR) is an advanced digital PCR technology developed to provide absolute quantitation of target DNA. In this study, we validated the clinical performance of ddPCR in determination of KRAS mutational status, and compared ddPCR results with those obtained by Sanger sequencing and peptide nucleic acid-clamping. Of 81 colorectal adenocarcinoma tissue samples, three repeated sets of KRASG12/G13 mutation were measured by ddPCR, yielding high consistency (ICC = 0.956). Receiver operating characteristic (ROC) curves were constructed to determine KRASG12/G13 mutational status based on mutant allele frequency generated by ddPCR. Using the best threshold cutoff (mutant allele frequency of 7.9%), ddPCR had superior diagnostic sensitivity (100%) and specificity (100%) relative to the two other techniques. Thus, ddPCR is effective for detecting the KRASG12/G13 mutation in colorectal adenocarcinoma tissue samples. By allowing definition of the optimal cutoff, ddPCR represents a potentially useful diagnostic tool that could improve diagnostic sensitivity and specificity.

Serum-Based KRASG12/G13 Mutation Detection Using Droplet Digital PCR: Clinical Implications and Limitations in Colorectal Adenocarcinoma With Tumor Heterogeneity.

BACKGROUND: Cell-free DNA (cfDNA) has arisen as an alternative target for evaluating somatic mutations in cancer. KRAS mutation status is critical for targeted therapy in colorectal adenocarcinoma (CRAC). We evaluated KRASG12/G13 mutations in cfDNA extracted from serum and compared the results with KRASG12/G13 mutations detected in tissue samples. We assessed the clinical significance of KRASG12/G13 mutation in serum in regard to recurrence and metastasis of CRAC. METHODS: A total of 146 CRAC patients were enrolled, and KRASG12/G13 mutations were evaluated in 146 pairs of serum and tissue samples. In addition, 35 pairs of primary and metastatic CRAC tissue samples were evaluated for KRASG12/G13 mutational status. RESULTS: Detection of KRASG12/13 mutation from serum and tissue had a 55% concordance rate, and serum detection had a sensitivity of 39.8%. Detection of the KRASG12/13 mutation yielded a 14% discordance rate between primary and metastatic tissue. CRAC patients with mutant KRASG12/13 mutation in serum but wild-type KRASG12/13 in tissue had concurrent KRASG12/13-mutant metastatic tumors, indicating spatial genetic heterogeneity. Changes in serum KRASG12/G13 mutation status during postoperative follow-up were associated with recurrence. Conclusion: Although serum detection of the KRASG12/13 mutation cannot substitute for detection in tissue, serum testing can support the interpretation of a CRAC patient's status in regard to concurrent metastasis. Dynamic changes in serum KRASG12/13 mutation status during follow-up indicated that cfDNA from serum represents a potential source for monitoring recurrence in CRAC patients.