Characterization of the complete mitochondrial genome of Takifugu pseudommus (Actinopterygii, Tetraodontidae).

The complete mitochondrial genome of Takifugu pseudommus was reported in the present study. It was 16,448 bp in length and consisted of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region. The nucleotide content of this genome was 29.91% for A, 29.09% for C, 15.20% for G, and 25.80% for T. The phylogenetic tree, constructed with complete mitochondrial genome, suggested T. pseudommus was closely related with T. chinensis, T. flavidus, and T. rubripes among Tetraodontidae species. This study could provide an important dataset for genetic diversity and species identification among the genus Takifugu.

Identification and Authentication of Commercial Mi-iuy Croaker (Miichthys miiuy) Products by Two PCR-Based Methods.

ABSTRACT: Mi-iuy croaker (Miichthys miiuy) is one of the most important ingredients of Korean cuisine and, thus, has a high economic value. However, the similar morphological traits among croaker fish belonging to family Sciaenidae are often exploited for seafood fraud. In this study, an M. miiuy-specific primer set was designed and further improved by the development of a rapid and cost-effective duplex PCR method. The specificity of M. miiuy-specific duplex PCR was tested using 22 seafood species, and no cross-reactivity was observed. The sensitivity of the PCR assay was found to be 0.1 ng/µL. For the first time, labeling compliance of 43 commercial mi-iuy croaker products was verified using both full DNA barcoding and M. miiuy-specific duplex PCR methods. For species identification, BOLDSYSTEMS and GenBank database were screened with the consensus sequences of each PCR product as a query. This identification result was further confirmed using the M. miiuy-specific duplex PCR method. The findings of this study revealed that principal species substituted were law croaker (Pseudotolithus senegallus, n = 4), bigeye croaker (Micropogonias megalops, n = 3), whitemouth croaker (Micropogonias furnieri, n = 1), and tigertoothed croaker (Otolithes ruber, n = 1). A significant percentage (21%) of mislabeling was present in commercial mi-iuy products sold on the South Korean market.

Development of four PCR-based methods to differentiate tilefish species (Branchiostegus japonicus and B. albus).

The red tilefish (Branchiostegus japonicus) is an important ingredient and fishery resource in South Korea. Branchiostegus japonicus-specific and tilefish primer sets were designed, and four PCR-based methods were developed to differentiate B. japonicus and B. albus species. The specificity of the conventional and quantitative real-time PCR (qPCR) developed was confirmed using twenty species, showing no cross-activity, and the limit of detection was 0.1-0.001 ng/µL. Their accuracy was validated using a forensically informative nucleotide sequencing method on forty-seven red tilefish products. These two methods were further improved to develop direct triplex PCR and ultra-fast qPCR for the on-site food analysis, which could complete the entire analytical procedure within either 90 or 30 min, while maintaining the same accuracy. Therefore, these four PCR methods can be efficiently customized in various analytical areas and conditions, including field analysis, rapid screening, quality control, and labeling compliance, required by food manufacturing industry and regulatory authorities.

Comparison of quantitative methods based on SYBR Green real-time qPCR to estimate pork meat adulteration in processed beef products.

Quantitative real-time PCR (qPCR) is a modern technique that has been widely used for the detection of species used in meat products. For obtaining accurate and reliable qPCR results, we assessed two common DNA quantification methods for isolated DNA and five quantification approaches for qPCR products. DNA dilution based on spectrofluorometric results showed better qPCR results than those based on spectrophotometry in terms of linear correlation, amplification efficiency, and linear dynamic range. Binary pork-beef mixtures were used to construct standard curves of SYBR Green-based qPCR products using five quantification approaches, and they were validated and compared using in-house pork models. 18S rRNA gene normalization methods showed better trueness (-11.79% to -6.73%) than that of methods using absolute and relative standard curves (-28.52% to -18.64%) in a model burger. These normalized reference methods successfully estimated the quantities of pork meat in the range of 100%-0.01% in commercial beef products.

A rapid real-time PCR method to differentiate between mottled skate (Beringraja pulchra) and other skate and ray species.

Skates and rays are commercially important fish in South Korea, and among them, Beringraja pulchra has the highest economic value. However, the similar morphological traits among skates and rays are often exploited for seafood fraud. Here, we designed both Beringraja pulchra-specific and skate-universal primer sets, capable of detecting short sequences in the cytochrome oxidase subunit I gene, and developed highly sensitive and reliable quantitative real-time PCR (qPCR) assays to differentiate between Beringraja pulchra and other skate and ray species. AΔCq method based on differences in the amplification efficiency was developed, validated, and then used to confirm the presence of Beringraja pulchra in twenty-six commercial skate products. The averageΔCq value obtained for other skate species (18.94 ±â€¯3.46) was significantly higher than that of Beringraja pulchra (1.18 ±â€¯0.15). For on-site applications, we developed an ultra-fast qPCR assay, allowing for completion of the entire analytical procedure within 30 min.

The complete chloroplast genome of Codonopsis lanceolata (Campanulaceae).

The complete chloroplast genome sequence of Codonopsis lanceolata was determined by next generation sequencing. The total length of chloroplast genome of C. lanceolata was 169,447 bp long, including a large single-copy (LSC) region of 85,253 bp, a small single-copy (SSC) region of 8060 bp, and a pair of identical inverted repeat regions (IRs) of 38,067 bp. A total of 110 genes was annotated, resulting in 79 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. The phylogenetic analysis of C. lanceolata with related chloroplast genome sequences in this study provided the taxonomical relationship of C. lanceolata in the genus Campanula.

The complete chloroplast genome of Caltha Palustris (Ranunculaceae).

The complete chloroplast genome sequence of Caltha palustris, a species of the Ranunculaceae family, was characterized from the de novo assembly of HiSeq (Illumina Co.) paired-end sequencing data. The chloroplast genome of C. palustris was 155,292 bp in length, with a large single-copy (LSC) region of 84,120 bp, a small single-copy (SSC) region of 18,342 bp, and a pair of identical inverted repeat regions (IRs) of 26,415 bp. The genome contained a total of 114 genes, including 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The phylogenetic analysis of C. palustris with 14 related species revealed the closest taxonomical relationship with Hydrastis canadensis in the Ranunculaceae family.

Expression efficiency of NAT2 haplotypes in a Korean population.

Since NAT2 single-nucleotide polymorphisms (SNPs) are responsible for the efficacy of arylamines and hydrazine drugs, defining the effects of these SNPs in various ethnicities is an important factor in the development of personalized medicine. In the present study, we examined the expression efficiency of NAT2 using promoter haplotypes identified in a Korean population. To construct NAT2 promoter haplotypes, seven NAT2 promoter SNPs (rs4646241, rs4646242, rs4646243, rs4646267, rs4345600, rs4271002 and rs4646246) were genotyped in a total of 192 Korean subjects. A luciferase assay was performed using the three commonest haplotypes to evaluate enzyme expression level of NAT2 promoter haplotypes. The most common haplotype (TACGAGG) showed the lowest enzyme expression level (0.72 ± 0.06 relative light units (RLU)/[ß-galactosidase]). The second (CGTAAGA) and third (TATAACA) commonest haplotypes showed intermediate and the highest enzyme expression level (0.99 ± 0.05 and 1.45 ± 0.11 RLU/[ß-galactosidase]), respectively. Haplotype comparison among populations showed that Asian populations had a high proportion of the haplotype for lowest enzyme expression. Haplotype frequencies of Caucasian and African ethnicities were markedly different from those of Korean ethnicity. Results from the present study should contribute to the expansion of our current understanding of the pharmacogenetics field.

Specific PCR assays to determine bovine, porcine, fish and plant origin of gelatin capsules of dietary supplements.

Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules.

Hearing among male firefighters: a comparison with hearing data from screened and unscreened male population.

We investigated whether hearing loss is associated with firefighting. We conducted cross-sectional study comparing hearing threshold levels (HTLs) of 912 male firefighters with two hearing databases obtained from an otologically normal male Korean population (KONP) and a non-industrial noise-exposed male Korean population (KNINEP), considering age and the main roles of firefighters. Firefighters' age-adjusted HTLs were significantly worse than those of KONP (prevalence ratio (PR)=5.29, P<0.001)but not different from those of KNINEP (PR=0.99, P=0.550). Rescuers (PR=1.005, P<0.001) had worse hearing than the KNINEP after age adjustment. Comparison of firefighters' HTLs (50th and 90th percentiles) with those of KONP and KNINEP by age and frequency showed that firefighters' HTLs had significant increases (poorer hearing) across most age groups and frequencies compared with KONP. Compared with KNINEP, firefighters' HTLs were worse in the younger age groups (<45 years) but not different in the older age groups (>45 years). In conclusion, the hearing thresholds of younger firefighters and rescuers were worse than expected by normal aging alone. Future research should include longitudinal studies to consider variable risk factors, such as military service, smoking, and so on.

Determination of DPYD enzyme activity in Korean population.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil. This study was performed to analyze the association between DPYD genetic variants and DPYD enzyme activity in the Korean population. METHODS: We screened the genetic variants and analyzed the enzyme activity in 73 healthy Korean subjects (69 men and 4 women; mean age, 22.6 years). Direct sequencing was conducted using the ABI 3730XL system, and enzyme activity was determined using high-performance liquid chromatography. RESULTS: A total of 83 genetic variants were observed. Among the identified genetic variants, 32 were polymorphic including 3 core and 11 novel genetic variants. Association analysis between each genetic variant and enzyme activity in Korean subjects showed that 2 novel genetic variants, -832 G>A and -131 C>A, induced a significant difference in enzyme activity (P < 0.05). CONCLUSIONS: To our knowledge, this is the first study that has examined the association between enzyme activity and DPYD genetic variants in the Korean population. In this study, we identified novel genetic variants that are associated with the enzyme activity. These findings will be valuable for further pharmacogenetic studies and especially useful for personalized medicine.

Metabolic engineering of a glycerol-oxidative pathway in Lactobacillus panis PM1 for utilization of bioethanol thin stillage: potential to produce platform chemicals from glycerol.

Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production.

Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1.

Thin stillage (TS) is a waste residue that remains after bioethanol production, and its disposal reflects the high costs of bioethanol production. Thus, the development of cost-effective ways to process TS is a pending issue in bioethanol plants. The aim of this study was to evaluate the utilization of TS for the production of the valuable chemical, 1,3-propanediol (1,3-PDO), by Lactobacillus panis PM1. Different fermentation parameters, including temperature, pH and strains [wild-type and a recombinant strain expressing a NADPH-dependent aldehyde reductase (YqhD) gene] were tested in batch and fed-batch cultivations. The highest 1,3-PDO concentration (12.85 g/L) and yield (0.84 g/g) were achieved by batch fermentation at pH-4.5/30 °C by the YqhD recombinant strain. Furthermore, pH-controlled batch fermentation reduced the total fermentation period, resulting in the maximal 1,3-PDO concentration of 16.23 g/L and yield of 0.72 g/g in TS without an expensive nutrient or nitrogen (e.g., yeast extract, beef extract, and peptone) supplementation. The addition of two trace elements, Mg(2+) and Mn(2+), in TS increased 1,3-PDO yield (0.74 g/g) without 3-hydroxypropionaldehyde production, the only intermediate of 1,3-PDO biosynthetic pathway in L. panis PM1. Our results suggest that L. panis PM1 can offer a cost-effective process that utilizes the TS to produce a value-added chemical, 1,3-PDO.

Transcriptional repressor role of PocR on the 1,3-propanediol biosynthetic pathway by Lactobacillus panis PM1.

PURPOSE OF WORK: The regulatory role of a transcriptional regulator (PocR) in the 1,3-propanediol biosynthetic pathway of Lactobacillus panis PM1 contributes to the optimization of 1,3-propanediol production by this strain, which potentially will lead to 1,3-propanediol manufacturing efficiencies. Lactobacillus panis PM1 can utilize a 1,3-propanediol (1,3-PDO) biosynthetic pathway, consisting of diol dehydratase (PduCDE) and 1,3-PDO dehydrogenase, as a NADH recycling system, to survive under various environmental conditions. In this study, we identified a key transcriptional repressor (PocR) which was annotated as a transcriptional factor of AraC family as part of the 1,3-PDO biosynthetic pathway of L. panis PM1. The over-expression of the PocR gene resulted in the significant repression (81 %) of pduC (PduCDE large subunit) transcription, and subsequently, the decreased activity of PduCDE by 22 %. As a result of the regulation of PduCDE, production of both 3-hydroxypropionaldehyde and 1,3-PDO in the PocR over-expressing strain were significantly decreased by 40 % relative to the control strain. These results clearly demonstrate the transcriptional repressor role of PocR in the 1,3-PDO biosynthetic pathway.

Regulation of dual glycolytic pathways for fructose metabolism in heterofermentative Lactobacillus panis PM1.

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli that use the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway as their central metabolic pathway and are reportedly unable to grow on fructose as a sole carbon source. We isolated a variant PM1 strain capable of sporadic growth on fructose medium and observed its distinctive characteristics of fructose metabolism. The end product pattern was different from what is expected in typical group III lactobacilli using the 6-PG/PK pathway (i.e., more lactate, less acetate, and no mannitol). In addition, in silico analysis revealed the presence of genes encoding most of critical enzymes in the Embden-Meyerhof (EM) pathway. These observations indicated that fructose was metabolized via two pathways. Fructose metabolism in the PM1 strain was influenced by the activities of two enzymes, triosephosphate isomerase (TPI) and glucose 6-phosphate isomerase (PGI). A lack of TPI resulted in the intracellular accumulation of dihydroxyacetone phosphate (DHAP) in PM1, the toxicity of which caused early growth cessation during fructose fermentation. The activity of PGI was enhanced by the presence of glyceraldehyde 3-phosphate (GAP), which allowed additional fructose to enter into the 6-PG/PK pathway to avoid toxicity by DHAP. Exogenous TPI gene expression shifted fructose metabolism from heterolactic to homolactic fermentation, indicating that TPI enabled the PM1 strain to mainly use the EM pathway for fructose fermentation. These findings clearly demonstrate that the balance in the accumulation of GAP and DHAP determines the fate of fructose metabolism and the activity of TPI plays a critical role during fructose fermentation via the EM pathway in L. panis PM1.

Contributions of citrate in redox potential maintenance and ATP production: metabolic pathways and their regulation in Lactobacillus panis PM1.

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli and can utilize various NADH-reoxidizing routes (e.g., citrate, glycerol, and oxygen) according to environmental conditions. In this study, we investigated the ability of L. panis PM1 to produce succinate, acetate, and lactate via citrate utilization. Possible pathways, as well as regulation, for citrate metabolism were examined on the basis of the genome sequence data and metabolic profiles of L. panis PM1. The presence of citrate led to the up-regulation, at the transcriptional level, of the genes encoding for citrate lyase, malate dehydrogenase, and malic enzyme of the citrate pathways by 10- to 120-fold. The transcriptional regulator of the dha operon coding for glycerol dehydratase of L. panis PM1 repressed the expression of the citrate lyase gene (10-fold). Metabolite analyses indicated that the transcriptional enhancement by citrate stimulated succinate yield. Citrate metabolism contributed to energy production by providing a major alternate pathway for NAD(+) regeneration and allowed acetyl phosphate to yield acetate/ATP instead of ethanol/NAD(+). Additionally, a branching pathway from oxaloacetate to pyruvate increased the pool of lactate, which was then used to produce ATP during stationary phase. However, the redirection of NADH-to-citrate utilization resulted in stress caused by end-products (i.e., succinate and acetate). This stress reduced succinate production by up to 50 % but did not cause significant changes at transcriptional level. Overall, citrate utilization was beneficial for the growth of L. panis PM1 by providing a NAD(+) regeneration route and producing extra ATP.

Assessment of noise measurements made with a continuous monitoring in time.

The World Health Organization has stated that hearing loss is one of the top 10 health problems worldwide and that noise-induced hearing loss is the leading occupational disease. This work evaluated the noise exposure levels of several job categories for 24-h periods over 7 days to determine the contribution of each microenvironment to total noise exposure. The noise exposure levels of 47 individuals were continuously measured using personal noise dosimeters in metropolitan Seoul, Korea. Participants ranged in age from 20 to 50 yr and represented eight occupational groups. Participants were asked to attach the noise dosimeters and complete a time-activity diary 24 h a day for 7 days. The average Leq 24 h,w among these individuals was 74 dBA, which ranged from 64 to 96 dBA. The average Leq 24 h,w was highest for Korean traditional music apprentices, followed by heavy equipment operators, firefighters, service workers, office workers, industrial hygienists, graduate and undergraduate students, and housewives (89, 77, 76, 76, 75, 71, 71, and 71 dBA, respectively, p < 0.001). 38 (80.9%) were exposed to noise levels greater than 70 dBA, which corresponds to the World Health Organization's exposure limit.

Influence of oxygen on NADH recycling and oxidative stress resistance systems in Lactobacillus panis PM1.

Lactobacillus panis strain PM1 is an obligatory heterofermentative and aerotolerant microorganism that also produces 1,3-propanediol from glycerol. This study investigated the metabolic responses of L. panis PM1 to oxidative stress under aerobic conditions. Growth under aerobic culture triggered an early entrance of L. panis PM1 into the stationary phase along with marked changes in end-product profiles. A ten-fold higher concentration of hydrogen peroxide was accumulated during aerobic culture compared to microaerobic culture. This H2O2 level was sufficient for the complete inhibition of L. panis PM1 cell growth, along with a significant reduction in end-products typically found during anaerobic growth. In silico analysis revealed that L. panis possessed two genes for NADH oxidase and NADH peroxidase, but their expression levels were not significantly affected by the presence of oxygen. Specific activities for these two enzymes were observed in crude extracts from L. panis PM1. Enzyme assays demonstrated that the majority of the H2O2 in the culture media was the product of NADH: H2O2 oxidase which was constitutively-active under both aerobic and microaerobic conditions; whereas, NADH peroxidase was positively-activated by the presence of oxygen and had a long induction time in contrast to NADH oxidase. These observations indicated that a coupled NADH oxidase - NADH peroxidase system was the main oxidative stress resistance mechanism in L. panis PM1, and was regulated by oxygen availability. Under aerobic conditions, NADH is mainly reoxidized by the NADH oxidase - peroxidase system rather than through the production of ethanol (or 1,3-propanediol or succinic acid production if glycerol or citric acid is available). This system helped L. panis PM1 directly use oxygen in its energy metabolism by producing extra ATP in contrast to homofermentative lactobacilli.

Alkaline conditions stimulate the production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways.

A novel Lactobacillus panis PM1 isolate was found to be capable of converting glycerol to 1,3-propanediol (1,3-PDO), an increasingly valuable commodity chemical. In this study the effects of various process parameters, including glucose and glycerol concentrations, inoculum size, temperature, aeration, pH, and carbon source were examined to determine the optimal conditions for the production of 1,3-PDO using a culture method simulating late log to early stationary phases. Inoculum size did not influence the production of 1,3-PDO, and temperature variance showed similar 1,3-PDO production between 25 and 37 °C under the examined conditions. Glycerol concentration and pH played a primary role in the final concentration of 1,3-PDO. The highest production occurred at 150-250 mM glycerol when 50 mM glucose was available. Alkaline initial conditions (pH 9-10) stimulated the production of 1,3-PDO which concurrently occurred with increased acetic acid production. Under these conditions, 213.6 mM of 1,3-PDO were produced from 300 mM glycerol (conversion efficiency was 71 %). These observations indicated that the production of 1,3-PDO was associated with the shift of the metabolic end-product ethanol to acetic acid, and that this shift resulted in an excess concentration of NADH available for the processing of glycerol to 1,3-PDO.

Isolation and characterization of novel 1,3-propanediol-producing Lactobacillus panis PM1 from bioethanol thin stillage.

Conversion of glycerol to 1,3-propanediol (1,3-PDO) is an attractive option to increase the economic efficiency of the biofuel industry. A bacterial strain that produced 1,3-PDO in the presence of glycerol was isolated from thin stillage, the fermentation residue of bioethanol production. This 1,3-PDO-producing organism was identified as Lactobacillus panis through biochemical characteristics and by 16S rRNA sequencing. Characterization of the L. panis strain hereafter designated as PM1 revealed it was an aerotolerant acidophilic anaerobe able to grow over a wide range of temperatures; tolerant to high concentrations of sodium chloride, ethanol, acetic acid, and lactic acid; and resistant to many common antibiotics. L. panis PM1 could utilize glucose, lactose, galactose, maltose, xylose, and arabinose, but could not grow on sucrose or fructose. Production of 1,3-PDO by L. panis PM1 occurred only when glucose was available as the carbon source in the absence of oxygen. These metabolic characteristics strongly suggested NADH recycling for glucose metabolism is achieved through 1,3-PDO production by this strain. These characteristics classified L. panis PM1 within the group III heterofermentative lactic acid bacteria, which includes the well-characterized 1,3-PDO-producing strain, Lactobacillus reuteri. Metabolite production profiles showed that L. panis PM1 produced considerable amounts of succinic acid (~11-12 mM) from normal MRS medium, which distinguishes this strain from L. reuteri strains.