Age-Related Decrease in Pellino-1 Expression Contributes to Osteoclast-Mediated Bone Loss.

Aging-related bone loss is driven by various biological factors, such as imbalanced bone metabolism from decreased osteoblast and increased osteoclast activities. Various transcriptional and post-transcriptional factors increase osteoclast activity with aging; however, studies regarding the post-translational regulators of osteoclast activity are still limited. The ubiquitin E3 ligase Pellino-1 is a well-known post-translational regulator of inflammation. However, how Pellino-1 expression regulation affects osteoclast differentiation remains unclear. This study determined that Pellino-1 levels are reduced in bone marrow monocytes (BMMs) from 40-week-old mice compared to 4-week-old mice. Interestingly, conditional Knockout (cKO) of Pellino-1 in 6-week-old mice resulted in decreased bone mass, reduced body size, and lower weight than in Pellino-1 floxed mice; however, these differences are not observed in 20-week-old mice. The increased number of tartrate-resistant acid phosphatase (TRAP)-positive cells and serum levels of C-terminal telopeptides of type I collagen, a marker of bone resorption, in 6-week-old Pellino-1 cKO mice implied a connection between Pellino-1 and the osteoclast population. Enhanced TRAP activity and upregulation of osteoclast genes in BMMs from the cKO mice indicate that Pellino-1 deletion affects osteoclast differentiation, leading to decreased bone mass and heightened osteoclast activity. Thus, targeting Pellino-1 could be a potential gene therapy for managing and preventing osteoporosis.

The addition of jogi, Micropogonias undulates, affects amino acid content in kimchi fermentation.

The effects of jogi (the fish Atlantic croaker, Micropogonias undulatus) on the production of physicochemical components, such as color, organic acids, and amino acids, in kimchi, a traditional fermented vegetable food of Korea, were determined. As fermentation progressed, the color change of jogi-added kimchi increased, but in comparison with that of the control group without jogi-added kimchi, was difficult to distinguish with the naked eye. Reducing sugar decreased in all experimental groups, and as fermentation progressed, kimchi with jogi showed a lower value. Acetic acid, citric acid, lactic acid, and ethanol, were highly produced in both types of kimchi, and above all, the jogi-baechu-kimchi group showed higher acetic acid and lactic acid contents than the control group. The increase and decrease of amino acids were similar in both types of kimchi. However, significantly, immediately after manufacture, the savory components aspartic acid and glutamic acid were detected higher than the control group. Subsequently, the fermentation tended to decrease as it progressed, but the content was higher than that of the control group. The above results show that jogi addition has a greater effect on the contents of amino acid, especially the savory component, than on the physicochemical components.

Current status of the novel food ingredient safety evaluation system.

Increasing demand for new foods, technological development, and vegan market growth have led to an increase in new food ingredients, so the need for safety assessment of these ingredients is important. Representative safety assessment systems are the Generally Recognized as Safe (GRAS) notification of the Food and Drug Administration in the USA and the novel food system of the European Food Safety Authority in the European Union. GRAS is a notification system for information on food ingredients, food additives and functional foods under the responsibility of the applicant, while the novel food system assesses the safety of food ingredients excluding food additives. In Korea, a safety evaluation system is established for temporary food ingredients, which includes food ingredients without a domestic intake history. However, safety assessment systems for novel foods from other countries and food ingredients produced by the application of new technology need to be improved.

Novel Starter Strain Enterococcus faecium DMEA09 from Traditional Korean Fermented Meju.

The Enterococcus faecium strain DMEA09 was previously isolated from traditional Korean fermented meju. The objective of the current study was to investigate the traits of E. faecium strain DMEA09 as a starter candidate, focusing on its safety and technological properties. Regarding its safety, the DMEA09 strain was found to be sensitive to nine antibiotics (ampicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, tylosin, and vancomycin) by showing lower minimum inhibitory concentrations (MICs) than the cut-off values suggested by the European Union Food Safety Authority for these nine antibiotics. However, its MIC value for clindamycin was twice as high as the cut-off value. A genomic analysis revealed that strain DMEA09 did not encode the acquired antibiotic resistance genes, including those for clindamycin. The DMEA09 strain did not show hemolysis as a result of analyzing α- and ß-hemolysis. It did not form biofilm either. A genomic analysis revealed that strain DMEA09 did not encode for any virulence factors including hemolysin. Most importantly, multilocus sequence typing revealed that the clonal group of strain DMEA09 was distinguished from clinical isolates. Regarding its technological properties, strain DMEA09 could grow in the presence of 6% salt. It showed protease activity when the salt concentration was 3%. It did not exhibit lipase activity. Its genome possessed 37 putative protease genes and salt-tolerance genes for survivability under salt conditions. Consequently, strain DMEA09 shows safe and technological properties as a new starter candidate. This was confirmed by genome analysis.

Staphylococcus equorum plasmid pKS1030-3 encodes auxiliary biofilm formation and trans-acting gene mobilization systems.

The foodborne bacterium Staphylococcus equorum strain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid of S. equorum KS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (the ica operon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding gene repB, a double-stranded origin of replication, and two single-stranded origins of replication. The ica operon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed in S. aureus RN4220, the ica operon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 of S. equorum strain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties of S. equorum KS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.

Overgrowth of long bone in rabbits by growth stimulation through metaphyseal hole creation.

Overgrowth of long bones was noted in pediatric patients who underwent anterior cruciate ligament reconstruction. Hyperaemia during creating a metaphyseal hole and the microinstability made by the drill hole may induce overgrowth. This study aimed to determine whether metaphyseal hole creation accelerates growth and increases bone length and compare the effects of growth stimulation between metaphyseal hole creation and periosteal resection. We selected 7- to 8-week-old male New Zealand white rabbits. Periosteal resection (N = 7) and metaphyseal hole creation (N = 7) were performed on the tibiae of skeletally immature rabbits. Seven additional sham controls were included as age-matched controls. In the metaphyseal hole group, the hole was made using a Steinman pin at the same level of periosteal resection, and the cancellous bone beneath the physis was removed by curettage. The vacant space in the metaphysis below the physis was filled with bone wax. Tibiae were collected 6 weeks after surgery. The operated tibia was longer in the metaphyseal hole group (10.43 ± 0.29 cm vs. 10.65 ± 0.35 cm, P = 0.002). Overgrowth was higher in the metaphyseal hole group (3.17 ± 1.16 mm) than in the sham group (- 0.17 ± 0.39 mm, P < 0.001). The overgrowth in the metaphyseal hole group was comparable to that in the periosteal resection group (2.23 ± 1.52 mm, P = 0.287). In rabbits, metaphyseal hole creation and interposition with bone wax can stimulate long bone overgrowth, and the amount of overgrowth is similar to that seen in periosteal resection.

Clinical application of protein-enhanced diet using mealworms in patients undergoing hepato-pancreato-biliary surgery.

OBJECTIVE: Patients with or without cancers who undergo major gastrointestinal surgery experience malnutrition owing to their catabolic status during the postoperative period. In this study, we evaluated the effect of the clinical application of protein-enhanced diet using mealworms in patients who underwent hepato-pancreato-biliary surgeries. METHODS: This study was designed as a prospective, two-armed, and double-blinded phase III study. The target number of enrolled patients was 216, and the patients were randomized on a 1:1 basis, either to the trial group (consuming mealworms) or to the control group (consuming grain powder). The primary endpoint was to examine the changes in body composition, including phase angle. For secondary outcomes, the activities of immune cells were evaluated using the patients' blood samples. RESULTS: No difference in the demographic characteristics of patients was observed. The ratio of the actual protein intake to the recommended daily intake in the trial group was significantly higher than that in the control group (110.03% vs. 98.80%, P = 0.023). In the data on body composition measured by InBody S-10 (Biospace, Seoul, South Korea), the ratios in body cell mass, fat free mass, muscle mass, and phase angle at the study endpoint compared with those at admission showed no statistically significant difference between the two groups. Immune cell analyses suggested that cytotoxic T cells in the trial group had higher activity than in the study group (1.192 vs. 0.974, P = 0.028). CONCLUSIONS: In this study, protein-enhanced diet using mealworms clinically improved the activity of immune cells. However, it did not significantly improve the patients' nutritional status after they experienced hepato-pancreato-biliary surgeries.

2-Methylisoborneol (2-MIB) Excretion by Pseudanabaena yagii under Low Temperature.

Outbreaks of 2-methylisoborneol (2-MIB) contamination in drinking water sources cause inconvenient odor issues in the water distribution system. In this study, microscopy-based isolation with physiological and molecular phylogenetic characterization were performed to investigate and characterize the 2-MIB odor producers that caused an odor problem in the freshwater system of the North Han River in the autumn of 2018. A benthic cyanobacterium was isolated from 2-MIB odor-issue freshwater samples and was found to be phylogenetically affiliated with Pseudanabaena yagii (99.66% sequence similarity), which was recorded in South Korea for the first time. The 2-MIB synthesis gene sequences from the odor-issue freshwater samples showed 100% similarity with those in the P. yagii strains. Protein sequences of 2-MIB synthase observed in the genome of the isolated strain showed structural and functional characteristics similar to those observed in other Pseudanabaena species. The 2-MIB production rate increased slowly during mat formation on the vessel wall; however, it rapidly increased after the temperature dropped. The 2-MIB gene was continuously expressed regardless of the temperature changes. These results suggest that the 2-MIB odor issue in the North Han River might be caused by the release of 2-MIB from the mat-forming P. yagii species in a low-temperature freshwater environment.

Complete Genome and Plasmid Sequences of Raphidiopsis curvispora Strain GIHE-G1, Isolated from a Drinking Water Reservoir in South Korea.

The complete genome and plasmid sequences of Raphidiopsis curvispora strain GIHE-G1, a coiled filamentous heterocyst-forming cyanobacterium isolated from a drinking water reservoir in South Korea, are reported here. The genome information is expected to improve understanding of this species.

Draft Genome Sequence of Putative 2-Methylisoborneol-Producing Pseudanabaena yagii Strain GIHE-NHR1, Isolated from the North Han River in South Korea.

The draft genome sequence of Pseudanabaena yagii GIHE-NHR1, a filamentous cyanobacterium, is reported here. Comparative genome analysis suggests that this strain can produce an odor-causing compound (2-methylisoborneol) in water. The genome information is expected to improve the understanding of the putative 2-methylisoborneol production by the bacterium.

Draft Genome Sequence of Raphidiopsis raciborskii Strain GIHE 2018, Isolated from a Shallow Freshwater Pond in South Korea.

The draft genome sequence of Raphidiopsis (Cylindrospermopsis) raciborskii strain GIHE 2018, a filamentous nitrogen-fixing and potentially harmful cyanobacterium, is reported here. This is the first strain isolated from a shallow freshwater pond in South Korea. This information is expected to improve our understanding of the phylogeography of this species.

DNA Markers to Discriminate Cannabis sativa L. 'Cheungsam' with Low Tetrahydrocannabinol (THC) Content from Other South Korea Cultivars Based on the Nucleotide Sequences of Tetrahydrocannabinolic Acid Synthase and Putative 3-Ketoacyl-CoA Synthase Genes.

Cannabis sativa L. has been utilized for a long time as a traditional herbal medicine in Korea. Dry fruits, achenes, each containing a single seed of Cannabis, are currently prescribed as Ma In (Cannabis Semen), a laxative. As each achene is enclosed by a bract, in which tetrahydrocannabinol (THC), the main psychological active compound in Cannabis is synthesized; achene is easily contaminated by THC from bract remnants. Therefore, it is safer to harvest achenes from Cannabis with a low THC content. Seeds of hemp, a low THC Cannabis, were recently classified as possible sources of new pharmacologically active compounds. Thus, a proper method to select appropriate Cannabis plants with low THC among cultivars in South Korea for medicinal purpose is necessary. As a result of cross-selection, Cannabis L. cultivar "Cheungsam" (CH) with the lowest THC content among cultivars cultivated in South Korea has been developed. In this study, we developed two DNA markers to reliably discriminate CH from other local cultivars with higher THC contents. We developed primer sets CHF3/CHR2 to amplify the 642 bp DNA marker of CH based on differences in the nucleotide sequences of the THCA synthase gene, which encodes a key enzyme in THC synthesis. We then developed a CHF1/CHR3 primer set to amplify the 401 bp DNA marker of CH based on the differences in both the content of very long chain fatty acids (VLCFs) and the sequence of the putative 3-ketoacyl-CoA synthase (KCS) gene encoding enzymes synthesizing VLCFs among local cultivars.

Antidepressant-Like and Neuroprotective Effects of Ethanol Extract from the Root Bark of Hibiscus syriacus L.

Hibiscus syriacus L. (Malvaceae) is an important ornamental shrub in horticulture and has been widely used as a medical material in Asia. The aim of this study was to assess the antidepressant and neuroprotective effects of a root bark extract of H. syriacus (HSR) and to investigate the underlying molecular mechanisms. Using an animal model of restraint stress, we investigated the effects of HSR on depressive-like behaviors and on the expression levels of serotonin, corticosterone, and neurotrophic factors in the brain. The mice were exposed to restraint stress for 2 h per day over a period of 3 weeks and orally treated with HSR (100, 200, or 400 mg/kg/day). We also examined the neuroprotective effect of HSR using corticosterone-treated human neuroblastoma SK-N-SH cells. The cells were incubated with the extract for 24 h, followed by corticosterone stimulation for 1 h, and then cell viability assay, cellular ATP assay, mitochondrial membrane potential (MMP) assay, cellular reactive oxygen species (ROS) assay, and western blotting were used to investigate the neuroprotective effects of HSR. Administration of HSR not only reduced the immobility times of the restraint-stressed mice in the forced swimming and tail suspension tests, but also significantly increased sucrose preference in the sucrose preference test. In addition, HSR significantly reduced the plasma levels of corticosterone and increased the brain levels of serotonin. The extract also increased the phosphorylation level of cyclic AMP response element-binding (CREB) protein and the expression level of brain-derived neurotrophic factor (BDNF). The in vitro assays showed that HSR pretreatment increased cell viability and ATP levels, recovered MMP, decreased ROS levels, and increased the expression of CREB and BDNF in corticosterone-induced neurotoxicity. Taken together, our data suggest that HSR may have the potential to control neuronal cell damage and depressive behaviors caused by chronic stress.

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on cellulose synthase A (CesA) genes in flax (Linum usitatissimum L. 'Nike') seedlings.

INTRODUCTION: Cellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages. OBJECTIVE: To identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development. METHODS: The presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. 'Nike') seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10-4 and 10-3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4-6-day-old flax seedlings. RESULTS: ACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC. CONCLUSION: Ethylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

Identification and monitoring of Korean medicines derived from Cinnamomum spp. by using ITS and DNA marker.

In this study, we identified and evaluated the genetic relationships among Cinnamomum plants, which are used in traditional medicine. We also attempted to monitor the distribution of traditional medicines derived from Cinnamomum cassia by using DNA barcoding and a species-specific DNA marker. Plants of the genus Cinnamomum, and in particular C. cassia, are commonly used as medicinal herbs in the form of Cinnamomi Ramulus, Cinnamomi Cortex, and Cassiae Cortex Interior. However, it is difficult to distinguish among different Cinnamomum species based on morphological features, and so to overcome this limitation, nucleotide sequences of the internal transcribed spacer (ITS) region of Cinnamomum DNA were determined and compared. On the basis of the discrepancy in determined ITS sequences, a 408-bp product, amplified by the primer pair CC F1/CC R3, was developed as a C. cassia-specific DNA marker. Using the developed DNA marker in combination with the ITS 2 nucleotide sequence, we monitored imported and commercially supplied medicinal products derived from Cinnamomum plants in markets in Korean, China, and Japan. The results revealed that most of the specimens monitored were derived from C. cassia.

Application of Partial Internal Transcribed Spacer Sequences for the Discrimination of Artemisia capillaris from Other Artemisia Species.

Several Artemisia species are used as herbal medicines including the dried aerial parts of Artemisia capillaris, which are used as Artemisiae Capillaris Herba (known as "Injinho" in Korean medicinal terminology and "Yin Chen Hao" in Chinese). In this study, we developed tools for distinguishing between A. capillaris and 11 other Artemisia species that grow and/or are cultured in China, Japan, and Korea. Based on partial nucleotide sequences in the internal transcribed spacer (ITS) that differ between the species, we designed primers to amplify a DNA marker for A. capillaris. In addition, to detect other Artemisia species that are contaminants of A. capillaris, we designed primers to amplify DNA markers of A. japonica, A. annua, A. apiacea, and A. anomala. Moreover, based on random amplified polymorphic DNA analysis, we confirmed that primers developed in a previous study could be used to identify Artemisia species that are sources of Artemisiae Argyi Folium and Artemisiae Iwayomogii Herba. By using these primers, we found that multiplex polymerase chain reaction (PCR) was a reliable tool to distinguish between A. capillaris and other Artemisia species and to identify other Artemisia species as contaminants of A. capillaris in a single PCR.

Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants.

Some plants in the genus Artemisia have been used for medicinal purposes. Among them, Artemisia iwayomogi, commonly referred to as "Haninjin," is one of the major medicinal materials used in traditional Korean medicine. By contrast, Artemisia capillaris and both Artemisia argyi and Artemisia princeps, referred to as "Injinho" and "Aeyup," respectively, are used to treat diseases different from those for which "Haninjin" is prescribed. Therefore, the development of a reliable method to differentiate each Artemisia herb is necessary. We found that a random amplified polymorphic DNA (RAPD) method can be used to efficiently discriminate a few Artemisia plants from one another. To improve the reliability of RAPD amplification, we designed primer sets based on the nucleotide sequences of RAPD products to amplify a sequence-characterized amplified region (SCAR) marker of A. iwayomogi. In addition, we designed two other primer sets to amplify SCAR markers of "Aeyup" (A. argyi and A. princeps) along with "Injinho" (A. capillaris) and Artemisia japonica, which are also traded in Korean herbal markets. Using these three primer sets, we developed a multiplex PCR method concurrently not only to discriminate A. iwayomogi from other Artemisia plants, but also to identify Artemisia plants using a single PCR process.

A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms.

This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.

Effect of nebulized bovine surfactant for experimental otitis media with effusion.

OBJECTIVES: In this study, we evaluated the efficacy of nebulized bovine pulmonary surfactant on experimentally induced otitis media with effusion (OME) in guinea pigs. METHODS: Twenty guinea pigs were divided into three groups. Four untreated animals served as normal controls. Experimental OME was established in both ears of the remaining 16 animals by a transbullar injection of 10 microL of Pseudomonas aeruginosa lipopolysaccharide in saline. Thereafter, the guinea pigs received nebulized phosphate buffered saline (n=8) or nebulized bovine pulmonary surfactant (n=8). Nebulization was given daily for 7 days. On day 8, all the animals' passive opening pressure (POP) of the Eustachian tube was measured and histopathological observations of the bulla were made by light microscopy. RESULTS: Nebulized bovine pulmonary surfactant significantly reduced the POP compared to that of saline nebulization. The bovine pulmonary surfactant improved the tubal patency and produced less histopathologcally-evident edematous bullar mucosa. CONCLUSION: Nebulization of bovine pulmonary surfactant plays an important role in treating otitis media with effusion in guinea pigs. Our results suggest that the chosen nebulized bovine pulmonary surfactant can be of good clinical benefit for treating OME in the future.