Optimization of RT-PCR methods for enterovirus detection in groundwater.

Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105-106 copy/mL for CV-B5 and 106-107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.

Comparison of enrichment methods for isolating Enterohemorrhagic Escherichia coli in kimchi.

This study was conducted to compare the efficiency of four enrichment methods of Enterohemorrhagic Escherichia coli by using the 16S rRNA amplicon sequencing and a predictive model. Four different methods (US FDA, ISO, Japan Food Hygiene Association and Korea Ministry of Food and Drug Safety) were used to enrich EHEC in kimchi inoculated with cocktails of EHEC strains (NCCP 13720, NCCP 13721, and NCCP 14134). The maximum growth rate (µmax) and lag phase duration (LPD) were compared using the Baranyi model, and 16S rRNA targeted sequencing was performed with samples at the end of the exponential phase. As a result, the µmax and LPD values of Baranyi model developed for the four enriched media ranged from 0.82 to 0.92 and from 2.35 to 2.68, respectively, suggesting that the growth of EHEC was similar in all four enrichment media. As for the relative abundance of the bacterial composition at the family level, Enterobacteriaceae was identified as the major component (>50%) in all four enriched media. The relative abundance of Enterobacteriaceae was highest (>90%) in the two enriched media with 20 mg/L novobiocin, demonstrating that significant growth of non-targeted bacteria takes place in enrichment broths utilizing <20 mg/L novobiocin or different antibiotics. In conclusion, this study suggests that all four enrichment broth are suitable for growing EHEC in kimchi and the use and concentration of antibiotics such as novobiocin in enrichment media may have a critical role in species diversity.

Evaluation of Hygiene Indicators and Sampling Plan for Detecting Microbial Contamination in Health Functional Foods.

ABSTRACT: This study aimed to monitor microbial contamination levels in a variety of health functional foods and to establish new microbial criteria. Indicator organisms (i.e., aerobic bacteria, coliform bacteria, and Escherichia coli) were monitored in 10 health functional food categories (743 items, 3,715 samples). The mean total aerobic counts of ginseng and Korean red ginseng were -0.35 and -0.74 log CFU/g; and the mean total coliform counts were -1.4 and -1.39 log CFU/g, respectively. In addition, the mean total coliform counts of fiber and protein products were -1.34 and -1.22 log CFU/g, respectively. However, no aerobic or coliform cells were detected in any other health functional food products (vitamins, minerals, probiotics, milk thistle extract, propolis, eicosapentaenoic acid, docosahexaenoic acid, or lutein products), and no E. coli was detected in any of the categories. These results can potentially be used to update the microbial criteria of the Health Functional Food Code.

Development and validation of a predictive model for pathogenic Escherichia coli in fresh-cut produce.

This study was performed to develop and validate a predictive growth model of pathogenic Escherichia coli to ensure the safety of fresh-cut produce. Samples were inoculated with a cocktail of seven E. coli strains of five pathotypes (EHEC, Enterohemorrhagic E. coli; ETEC, Enterotoxigenic E. coli; EPEC, Enteropathogenic E. coli; EIEC, Enteroinvasive E. coli, and EAEC, Enteroaggregative E. coli) and stored at 4, 10, 12, 15, 25, 30, and 37°C. Growth of pathogenic E. coli was observed above 12°C. The primary growth model for pathogenic E. coli in fresh-cut produce was developed based on the Baranyi model. The secondary model was developed as a function of temperature for lag phase duration (LPD) and maximum specific growth rate (µmax) based on the polynomial second-order model. The primary and secondary models for pathogenic E. coli were fitted with a high degree of goodness of fit (R2 ≥ 0.99). The bias factor (Bf), accuracy factor (Af), and root mean square error (RMSE) were 0.995, 1.011, and 0.084, respectively. The growth model we developed can provide useful data for assessing the quantitative microbial risk of pathogenic E. coli in fresh-cut produce intended for human consumption. In addition, it is thought to be widely available in industries that produce, process, distribute, and sell fresh-cut produce.

Quantitative microbial risk assessment of pathogenic Escherichia coli in commercial kimchi in South Korea.

Owing to convenience, ease of preparation, and price, the consumption of commercial kimchi is gradually rising in South Korea. Here, we estimated the risk level posed by pathogenic Escherichia coli in commercial kimchi products using the quantitative microbial risk assessment (QMRA) approach to develop measures for preventing potential foodborne outbreaks from kimchi consumption. We collected 610 samples of commercial kimchi products produced in Korea, 267 kimchi samples from foreign countries imported to Korea, and 187 raw materials used in kimchi preparation, and analyzed them for contamination with pathogenic E. coli. A Predictive model was developed to observe the survival characteristics of pathogenic E. coli. A dose-response model was selected, and the risk level was estimated using @RISK software. Although a prior epidemiological study indicated the frequent occurrence of foodborne outbreaks arising from contaminated kimchi products consumed in food service facilities, we found a low probability of foodborne illness caused by pathogenic E. coli in commercial kimchi products.

Biosynthesis of (R)-(-)-1-Octen-3-ol in Recombinant Saccharomyces cerevisiae with Lipoxygenase-1 and Hydroperoxide Lyase Genes from Tricholoma matsutake.

Tricholoma matsutake is an ectomycorrhizal fungus, related with the host of Pinus densiflora. Most of studies on T. matsutake have focused on mycelial growth, genes and genomics, phylogenetics, symbiosis, and immune activity of this strain. T. matsutake is known for its unique fragrance in Eastern Asia. The most major component of its scent is (R)-(-)-1-octen-3-ol and is biosynthesized from the substrate linoleic acid by the sequential reaction of lipoxygenase and peroxide lyase. Here, we report for the first time the biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake using the yeast Saccharomyces cerevisiae as a host. In this study, cDNA genes correlated with these reactions were cloned from T. matsutake, and expression studies of theses genes were carried out in the yeast Saccharomyces cerevisiae. The product of these genes expression study was carried out with Western blotting. The biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake in recombinant Saccharomyces cerevisiae was subsequently identified with GC-MS chromatography analysis. The biosynthesis of (R)-(-)-1-octen-3-ol with S. cerevisiae represents a significant step forward.

Comamonas flocculans sp. nov., a Floc-Forming Bacterium Isolated from Livestock Wastewater.

A Gram-negative, aerobic, non-motile, rod-shaped, floc-forming, and non-spore-forming bacterium, designated as NLF-7-7T, was isolated from the biofilm of a sample collected from a livestock wastewater treatment plant in Nonsan, Republic of Korea. Strain NLF-7-7T, forms a visible floc and grows in the flocculated state. Cells of strain NLF-7-7T grew optimally at pH 6.5 and 30 °C and in the presence of 0.5% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NLF-7-7T belonged to the family Comamonadaceae, and was most closely related to Comamonas badia DSM 17552T (95.8% similarity) and Comamonas nitrativorans 23310T (94.0% similarity). The phylogenetic and phenotypic data indicate strain NLF-7-7T is clearly distinguished from the Comamonas lineage. The major cellular fatty acids were C10:0 3OH, C16:0, and summed feature 3 (C16:1 ω6c/C16:1 ω7c). The respiratory quinone was Q-8. The polar lipids were composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminolipid. The DNA G+C content of strain NLF-7-7 was 68.0 mol%. Based on the phenotypic, chemotaxonomic, and phylogenetic properties, strain NLF-7-7T represents a novel species of the genus Comamonas, for which the name Comamonas flocculans sp. nov. is proposed. The type strain is C. flocculans NLF-7-7T (=KCTC 62943T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Comamonas flocculans NLF-7-7T is MN527436. The whole-genome shotgun BioProject Number is PRJNA555370 with the Accession Number CP042344.

Noncovalent tethering of nucleic acid aptamer on DNA nanostructure for targeted photo/chemo/gene therapies.

Here, we report various therapeutic cargo-loadable DNA nanostructures that are shelled in polydopamine and noncovalently tethered with cancer cell-targeting DNA aptamers. Initial DNA nanostructure was formed by rolling-circle amplification and condensation with Mu peptides. This DNA nanostructure was loaded with an antisense oligonucleotide, a photosensitizer, or an anticancer chemotherapeutic drug. Each therapeutic agent-loaded DNA nanostructure was then shelled with polydopamine (PDA), and noncovalently decorated with a poly adenine-tailed nucleic acid aptamer (PA) specific for PTK7 receptor, resulting in PA-tethered and PDA-shelled DNA nanostructure (PA/PDN). PDA coating shell enabled photothermal therapy. In the cells overexpressing PTK7 receptor, photosensitizer-loaded PA/PDN showed greater photodynamic activity. Doxorubicin-loaded PA/PDN exerted higher anticancer activity than the other groups. Antisense oligonucleotide-loaded PA/PDN provided selective reduction of target proteins compared with other groups. Our results suggest that the PA-tethered and PDA-shelled DNA nanostructures could enable the specific receptor-targeted phototherapy, chemotherapy, and gene therapy against cancer cells.

Genetic and phenotypic characterization of the novel mouse substrain C57BL/6N Korl with increased body weight.

In inbred mouse lines, there is generally little genetic difference between individuals. This small genetic variability facilitates carrying out research on minute changes of various traits and the gene pool. Also, characterizing the diversity and detecting selective genetic and phenotypic signatures are crucial to understanding the genomic basis of a population and to identify specific patterns of evolutionary change. In this study, we investigated the underlying genetic profiles of a newly developed mouse strain, C57BL/6NKorl (Korl), established through sibling mating over 30 generations. To analyse the distinctive genomic features of Korl mice, we used whole-genome sequencing from six samples, which were compared to those of other C57BL/6N-based mouse strains. Korl strain-specific polymorphisms were identified and signatures of a selective sweep were detected. In particular, the candidate genes related to the increased body weight of the Korl strain were identified. Establishment of the genetic profile of Korl mice can provide insight into the inbreeding-induced changes to the gene pool, and help to establish this strain as a useful model for practical and targeted research purposes.

Claudin 4-targeted nanographene phototherapy using a Clostridium perfringens enterotoxin peptide-photosensitizer conjugate.

In this study we designed a claudin 4-directed dual photodynamic and photothermal system, in which a 30-amino acid claudin 4-binding peptide, Clostridium perfringens enterotoxin (CPE), was linked to a photodynamic agent chlorin e6 (Ce6) through a polyethylene glycol spacer (CPC) and anchored onto reduced graphene oxide (rGO) nanosheets to form CPC/rGO nanosheets. For comparison, a conjugate of polyethylene glycol and Ce6 (PC) was anchored onto the rGO nanosheets to generate PC/rGO. Both PC and CPC generated reactive oxygen species upon irradiation at 660 nm. Application of CPC/rGO to claudin 4-overexpressing U87 glioblastoma cells in vitro resulted in a significantly higher cellular uptake compared to application of PC/rGO. Upon irradiation at 660 and 808 nm, the CPC/rGO-treated U87 cells generated significantly higher reactive oxygen species and caused significantly higher temperature increase, and showed most potent anticancer effect compared to the other groups. Taken together, these results suggest that CPC/rGO is potentially useful as a tumor-specific combined phototherapy.

Comparison of commonly used ICR stocks and the characterization of Korl:ICR.

Mouse is a commonly used animal in life science studies and is classified as outbred if genetically diverse and inbred if genetically homogeneous. Outbred mouse stocks, are used in toxicology, oncology, infection and pharmacology research. The National Institute of Food and Drug Safety Evaluation (NIFDS; former the Korea National Institute of Health) have bred ICR mice for more than 50 years. We investigated to provide users with information and promote accountability to the Korl:ICR. To secure the indigenous data, biological characteristics of Korl:ICR were identified by comparing with other ICR stocks. This domestic ICR stock was denominated as 'Korl:ICR'. Phylogenetic analysis using SNPs indicated that the population stratification of the Korl:ICR was allocated different area with other ICR. In addition, we measured litter size, body weight, body length, various organ weight, hematology and clinical blood chemistry of the Korl:ICR compared to other ICR. Otherwise, there are no significant differences among the biological phenotypes of Korl:ICR and other ICR. These results suggest that as a genetically indigenous source colony, the Korl:ICR is seperated (or independent) stock with other ICR. Also, we confirmed that there is no difference among the Korl:ICR and other ICR on biological phenotypes. Therefore, the Korl:ICR source colony might be a new stock in distinction from other ICR, it is a good milestone in securing ownership of the national laboratory animal resource. The NIFDS expects that the Korl:ICR mice will be useful animal resource for our domestic researchers.

Wiryeongtang regulates hypertonicity-induced expression of aquaporin-2 water channels in mIMCD-3 cells.

The kidneys have a key role in the homeostasis of water excretion and reabsorption. Water channels, particularly aquaporin-2 (AQP2), are important proteins in water homeostasis in the body through the short­term and long-term regulation of water permeability. Wiryeongtang (WRT) is a well-known traditional oriental medicine, which is used for the treatment of chronic edema and dysuresia. The aim of the present study was to evaluate the inhibitory effect of WRT on the hypertonicity-induced expression of AQP2 in the inner medullary collecting duct cell line (IMCD­3). Western blotting, reverse transcription­polymerase chain reaction and immunofluorescence analysis were performed to determine the effect of WRT under hypertonic stress. WRT attenuated the 175 mM NaCl hypertonic stress­induced increases in protein and mRNA levels of AQP2 and apical membrane insertion in a concentration­dependent manner. However, no differences were observed in the levels of AQP1, AQP3 or AQP4 between the hypertonic stress and WRT groups. WRT attenuated the hypertonicity-induced phosphorylation of glucocorticoid-inducible protein kinase 1. In addition, the mRNA expression of tonicity­responsive enhancer binding protein was attenuated by WRT under hypertonic stress. Pretreatment with WRT also decreased the hypertonic stress­induced expression of AQP2, as with KT5720, a protein kinase A inhibitor. These results provided evidence of the beneficial effect of the traditional formula WRT in regulating water balance in hypertonic stress of the renal collecting ducts.

Nanoformulation-based sequential combination cancer therapy.

Although combining two or more treatments is regarded as an indispensable approach for effectively treating cancer, the traditional cocktail-based combination therapies are seriously limited by coordination issues that fail to account for differences in the pharmacokinetics and action sites of each drug. The careful manipulation of dosing regimens, such as by the sequential application of combination treatments, may satisfy the temporal and spatial needs of each drug and achieve successful combination antitumor therapy. Nanotechnology-based carriers might be the best tools for sequential combination therapy, as they can be loaded with multiple cargos and may provide targeted and sustained delivery to target tumor cells. Single nanoformulations capable of sequentially releasing drugs have shown synergistic anticancer activity, such as by sensitizing tumor cells through cascaded drug delivery or remodeling the tumor vasculature and microenvironment to enhance the tumor distribution of nanotherapeutics. This review highlights the use of nanotechnology-based multistage drug delivery for cancer treatment, focusing on the ability of such formulations to enhance antitumor efficacy by applying sequential treatment and modulating dosing regimens, which are challenges currently being faced in the clinic.

Activation of AMPK by berberine induces hepatic lipid accumulation by upregulation of fatty acid translocase CD36 in mice.

Emerging evidence has shown that berberine has a protective effect against metabolic syndrome such as obesity and type II diabetes mellitus by activating AMP-activated protein kinase (AMPK). AMPK induces CD36 trafficking to the sarcolemma for fatty acid uptake and oxidation in contracting muscle. However, little is known about the effects of AMPK on CD36 regulation in the liver. We investigated whether AMPK activation by berberine affects CD36 expression and fatty acid uptake in hepatocytes and whether it is linked to hepatic lipid accumulation. Activation of AMPK by berberine or transduction with adenoviral vectors encoding constitutively active AMPK in HepG2 and mouse primary hepatocytes increased the expression and membrane translocation of CD36, resulting in enhanced fatty acid uptake and lipid accumulation as determined by BODIPY-C16 and Nile red fluorescence, respectively. Activation of AMPK by berberine induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and subsequently induced CCAAT/enhancer-binding protein ß (C/EBPß) binding to the C/EBP-response element in the CD36 promoter in hepatocytes. In addition, hepatic CD36 expression and triglyceride levels were increased in normal diet-fed mice treated with berberine, but completely prevented when hepatic CD36 was silenced with adenovirus containing CD36-specific shRNA. Taken together, prolonged activation of AMPK by berberine increased CD36 expression in hepatocytes, resulting in fatty acid uptake via processes linked to hepatocellular lipid accumulation and fatty liver.

Progress of Middle East respiratory syndrome coronavirus vaccines: a patent review.

INTRODUCTION: Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged as a new pathogen, causing severe complications and a high case fatality rate. No direct treatments are available as yet, highlighting the importance of prevention through suitable vaccination regimes. The viral spike (S) protein has been characterized as a key target antigen for vaccines. In particular, S protein domains have been utilized to produce high titers of neutralizing antibodies. Areas covered: Since the first report of MERS-CoV infection, a limited number of MERS-CoV-specific patents have been filed. Patents related to MERS-CoV are categorized into three areas: treatments, antibodies, and vaccines (receptor-related). This review mainly focuses on the types and efficacies of vaccines, briefly covering treatments and antibodies against the virus. MERS-CoV vaccine forms and delivery systems, together with comparable development strategies against SARS-CoV are additionally addressed. Expert opinion: Vaccines must be combined with delivery systems, administration routes, and adjuvants to maximize T-cell responses as well as neutralizing antibody production. High immune responses require further study in animal models, such as human receptor-expressing mice, non-human primates, and camels. Such a consideration of integrated actions should contribute to the rapid development of vaccines against MERS-CoV and related coronaviruses.

Selective Activation of Anticancer Chemotherapy by Cancer-Associated Fibroblasts in the Tumor Microenvironment.

Background: The tumor microenvironment has recently emerged as a new target of anticancer chemotherapy. Selective activation of anticancer chemotherapy in the tumor microenvironment would further reduce the toxicity of anticancer drugs toward normal tissues. Fibroblast activation protein (FAP) is known to be selectively overexpressed on cancer-associated fibroblasts (CAFs) in the tumor microenvironment. Here, we designed an anticancer chemotherapeutic system based on promelittin, a peptide toxin that is selectively converted from an inactive form to the pore-forming melittin upon cleavage by FAP in the tumor microenvironment. Methods: We conjugated promelittin-containing FAP-cleavable sequences to pegylated phospholipids and anchored them to reduced graphene oxide (rGO) nanosheets. The resulting nanosheets, PL-rGO, were tested for hemolysis and used for doxorubicin delivery. In vitro cocultures and in vivo tumor growth (n = 5 mice per group) with tissue immunostaining were used to test the selective activation of anticancer chemotherapy by FAP expressed on CAFs. Results: FAP-specific hemolytic activity of PL-rGO was observed in cocultures of CAFs and HT29 cells but not in HT29 cells alone. Doxorubicin-loaded PL-rGO (Dox/PL-rGO) showed 3.4-fold greater cell-killing efficacy (compared with free Dox in the CAF/HT29 coculture system, effects that were not observed in HT29 cells alone). Intravenously administered Dox/PL-rGO reduced the growth of HT29 tumors more effectively than other treatments (Dox/PL-rGO: mean = 200.6 mm(3), 95% confidence interval [CI] = 148.7 to 252.5 mm(3); free Dox: mean = 697.0 mm(3), 95% CI = 646.9 to 747.1 mm(3), PL: mean = 565.0 mm(3), 95% CI = 550.5 to 579.6 mm(3); Dox/rGO: mean = 637.6 mm(3), 95% CI = 619.5 to 655.7 mm(3); PL-rGO: mean = 464.4 mm(3), 95% CI = 433.0 to 495.8 mm(3)). Immunostaining of tumor tissues revealed that survival of CAFs and HT29 cells was lowest in the group treated with Dox/PL-rGO. Conclusions: The demonstration of selective activation of PL-rGO by FAP on CAFs suggests that PL-rGO may serve as a tumor microenvironment-responsive anticancer chemotherapy system.

Graphene-based nanosheets for delivery of chemotherapeutics and biological drugs.

Graphene-based nanosheets (GNS), including graphenes, graphene oxides and reduced graphene oxides, have properties suitable for delivery of various molecules. With their two-dimensional structures, GNS provide relatively high surface areas and capacity for non-covalent π-π stacking and hydrophobic interactions with various drug molecules. Currently, GNS-based delivery applications extend to chemotherapeutics as well as biological drugs, including nucleic acid drugs, proteins, and peptides. Surfaces of GNS have been modified with various polymers, such as polyethylene glycol and biopolymers, which enhance biocompatibility and increase drug loading. Anticancer drugs are prominent among chemotherapeutic agents tested, and have been loaded onto GNS with relatively high loading capacities compared with other nanocarriers. For enhanced distribution to specific tissues, GNS have been covalently or non-covalently modified with targeting ligands, including folic acid, transferrins, and others. In this review, we cover the current status of GNS for delivery of anticancer chemotherapeutics and biological drugs, with a focus on nucleic acid drugs. Remaining challenges for the application of GNS for drug-delivery systems and future perspectives are also addressed.

Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.

Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy.

Biomimetic DNA nanoballs for oligonucleotide delivery.

Here, we designed biomimetic DNA nanoballs for delivery of multiple antisense oligonucleotides (ASOs). DNA templates with ASOs-complementary sequences were amplified by rolling circle amplification (RCA). RCA products were loaded with two types of ASOs by hybridization, condensed using adenovirus-derived Mu peptide, and coated with hyaluronic acid (HA) for delivery into CD44-overexpressing tumor cells. HA-coated, Mu peptide-condensed, dual ASO-loaded DNA nanoballs (HMA nanoballs) showed considerable cellular entry of Cy5-incorporated RCA product DNA and fluorescent ASOs, whereas Mu peptide-condensed, dual ASO-loaded DNA nanoballs (MA nanoballs) revealed limited uptake. Dual ASOs, Dz13 and OGX-427, delivered by HMA nanoballs could reduce the levels of protein targets and exert anticancer effects. Enhanced tumor distribution was observed for fluorescent HMA nanoballs than the corresponding MA nanoballs. Upon intravenous co-administration with doxorubicin, HMA nanoballs exerted the greatest anti-tumor effects among the groups. These results suggest HMA nanoballs as a nanoplatform for sequence-specific delivery of multiple ASOs and other functional oligonucleotides.