Search

Objective: We developed a simple and validated liquid chromatography tandem mass spectrometry( LC-MS/MS) for quantification of etodolac using pioglitazone as an internal standard (IS) to assess pharmacokinetics and to appraise bioequivalence of two formulations of etodolac (reference and tested) in 27 healthy Korean subjects. Methods: Isocratic mobile phase consisted of 10 mM ammonium formate and acetonitrile were used to separate the analytes on a Gemini C18 column. Also, analytes were analyzed by MS/MS in multiple reaction monitoring (MRM) mode using the transitions of (M+H)+ ions, m/z 288.2→ ; 172.3 and m/z 357.1→ 134.2 for quantification of etodolac and IS each. The standard calibration curves displayed significant linearity within the range of 0.2-30.0 μ g/mL (r2=0.9956, 1/x2 weighting) with LLOQ of 0.1 μg/mL. Results: The retention times of etodolac and the IS were 0.77 min and 0.57 min each, indicating the high-throughput potential of the proposed method. The pharmacokinetic parameters were calculated from the plasma samples and data form the reference and test drugs were represented as follows; Area under plasma concentration-time curve (AUCt) (78.03 vs. 84.00 μgxh/mL), AUC∞ (86.67 vs. 93.92 μgxh/mL), maximal plasma concentration (Cmax) (19.49 vs. 18.94 μg/mL), time for maximal concentrations (Tmax) (2.13 vs. 2.26 h), Plasma elimination half-life (T1/2) (8.12 vs. 8.47 h), elimination rate constant (λz) (0.0853 vs. 0.0818 h-1). Pharmacokinetic parameters with 90% confidence interval fall within the bioequivalence range of 80-125%. Conclusion: Thus, the new testified method was successfully applied for the pharmacokinetic and bioequivalence studies for two etodolac formulations.

We developed the LC-MS/MS method through a comparison of the pharmacokinetic characteristics of imidapril to assess the bioequivalence of two types of imidapril in 31 normal Korean subjects. Blood sample were collected at 13 time points for 12 h after dosing and detected by LC-MS/MS in the range of 0.2-100 ng/mL with LLOQ of 0.2 ng/mL. Pharmacokinetic parameters analyzed from the plasma samples and data from the reference and test drugs in the plasma were represented such as AUC0-t (142.31 vs 131.95 ng•h/mL), AUC0-infinity (143.85 vs 133.72 ng•h/mL), Cmax(33.28 vs 30.46 ng/mL), Tmax(2.0 vs 1.9 h), half-life(1.6 vs 1.6 h), extrapolation (1.35 vs 1.46%), and Ke (0.46 vs 0.45 h-1). Pharmacokinetic parameters with a 90% confidence interval (CI) were 0.873–1.084 for AUC0-t and 0.863–1.149 for Cmax. Pharmacokinetic parameters with 90% CI were included within the bioequivalence range of 80–125% of the KFDA guidelines. Therefore, the two imidapril types were found to be bioequivalent during the fasting state in normal subjects.

A simple LC-MS/MS method has been advanced for measuring ciprofloxacin, using liquidliquid extraction of plasma with ether and methylene chloride (7/3, v/v) using ofloxacin as an internal standard (IS). The analytes were detected by HPLC using an isocratic mobile phase composed of methanol and 5 mM aqueous ammonium acetate (pH 3.0, 35/65, v/v) on a Luna phenyl-hexyl (Phenomenex 2.0 x 50 mm, 5 μm) column. The eluate was analyzed by MS/MS in the MRM mode using the transitions of the respective (M+H)+ ions (m/z 332.1→231.1 and m/z 362.1→261.1) for determination of ciprofloxacin and IS, respectively. The standard calibration curves indicated good linearity within the range of 0.05-5.0 μg/ml (r2=0.997, 1/x2 weighting). The lower limit of quantification (LLOQ) was 0.05 μg/ml. The retention times of ciprofloxacin and IS were 1.79 and 1.51 minutes, respectively. In addition, no significant impurities were found that interfered with the analysis. Acceptable precision and accuracy were obtained for the concentration range of the standard curve. The validated method was successfully applied to bioequivalence and pharmacokinetic studies of two formulations of ciprofloxacin (single 250 mg doses) in 24 healthy Korean volunteers.

The molecular mechanism of biofilm formation by SalmonellaTyphimuriun DT104 was characterized for a better understanding of its attachment and colonization in food processing environments. A library of random mutagenized clones was screened for phenotypic analyses of their ability to form biofilm, pellicle, curli, and cellulose. The genes identified were involved in lipopolysaccharide synthesis, assembly of flagella, regulation of rRNA biosynthesis, and outer membrane transportation and signaling. The insertion of transposon in flgK, rfbA, nusB, and pnpgenes resulted in decreased biofilm formation. Alterations of flagellar and lipopolysaccharide production were confirmed in the flgKmutant and rfbAmutant, respectively. Biofilm formation by these four mutants in meat and poultry broths and their attachment on surfaces of stainless steel and glass were significantly reduced compared with those of the wild-type strain (P< 0.05). On the contrary, the mutation of STM4263 and yjcCgenes in SalmonellaTyphimuriun DT104 resulted in increased biofilm formation and attachment of the species in tested broths and on contact surfaces. Our findings suggest that many factors, such as production of exopolymeric substances and their efficient transportation through outer membrane, expression of flagella, and regulation of exoribonucleases and RNA-binding protein, could be involved in biofilm formation and attachment of SalmonellaTyphimurium DT104 on contact surfaces.

The biofilm-forming capability of Salmonella entericaserotypes Typhimurium and Heidelberg, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coliO157:H7, Klebsiella pneumoniae, and Acinetobacter baumanniiisolated from humans, animal farms, and retail meat products was evaluated by using a microplate assay. The tested bacterial species showed interstrain variation in their capabilities to form biofilms. Strong biofilm-forming strains of S. entericaserotypes, E. coliO157: H7, P. aeruginosa, K. pneumoniae, and A. baumanniiwere resistant to at least four of the tested antibiotics. To understand their potential in forming biofilms in food-processing environments, the strong biofilm formers grown in beef, turkey, and lettuce broths were further investigated on stainless steel and glass surfaces. Among the tested strains, SalmonellaTyphimurium phage type DT104 (SalmonellaTyphimurium DT104) isolated from retail beef formed the strongest biofilm on stainless steel and glass in beef and turkey broths. K. pneumoniae, L. monocytogenes, and P. aeruginosawere also able to form strong biofilms on the tested surface materials. SalmonellaTyphimurium DT104 developed a biofilm on stainless steel in beef and turkey broths through (i) initial attachment to the surface, (ii) formation of microcolonies, and (iii) biofilm maturation. These findings indicated that SalmonellaTyphimurium DT104 along with other bacterial pathogens could be a source of cross-contamination during handling and processing of food.

Multidrug-resistant enteric bacteria were isolated from turkey, cattle, and chicken farms and retail meat products in Oklahoma. Among the isolated species, multidrug-resistant Klebsiella pneumoniaewas prevalently isolated from most of the collected samples. Therefore, a total of 132 isolates of K. pneumoniaewere characterized to understand their potential roles in the dissemination of antibiotic-resistance genes in the food chains. Multidrug-resistant K. pneumoniaewas most frequently recovered from a turkey farm and ground turkey products among the tested samples. All isolates were resistant to ampicillin, tetracycline, streptomycin, gentamycin, and kanamycin. Class 1 integrons located in plasmids were identified as a common carrier of the aadA1gene, encoding resistance to streptomycin and spectinomycin. Production of β-lactamase in the K. pneumoniaeisolates played a major role in the resistance to β-lactam agents. Most isolates (96%) possessed blaSHV-1. Five strains were able to express both SHV-11 (pI 6.2) and TEM-1 (pI 5.2) β-lactamase. Transfer of these antibiotic-resistance genes to Escherichia coliwas demonstrated by transconjugation. The bacterial genomic DNA restriction patterns by pulsed-field gel electrophoresis showed that the same clones of multidrug-resistant K. pneumoniaeremained in feathers, feed, feces, and drinking water in turkey environments, indicating the possible dissemination of antibiotic-resistance genes in the ecosystem and cross-contamination of antibiotic-resistant bacteria during processing and distribution of products.

An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130°C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90°C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130°C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.

Sixty-four multidrug-resistant isolates of Proteus mirabiliswere collected from retail meat products in Oklahoma. The isolates showed four different patterns of antibiotic resistance based on their resistant phenotype and genotypes. Most of these isolates were resistant to ampicillin, tetracycline, gentamycin, and kanamycin. Class 1 integrons were detected as a common carrier of the antibiotic-resistant genes, such as aadA1, aadB, and aadA2. A few isolates (9%) contained class 2 integrons with three gene cassettes included: dhfr1, sat1, and aadA1. These isolates were even resistant to nalidixic acid due to mutations in gyrAand parC. All ampicillin-resistant isolates contained blaTEM-1. Plasmids that contained class 1 or 2 integrons and blaTEM-1 were able to be transferred from P. mirabilisisolates into Escherichia coliby conjugation, indicating that conjugal transfer could contribute to the dissemination of antibiotic resistance genes between the Enterobacteriaceaespecies.

A polymerase chain reaction (PCR) assay for the rapid and sensitive detection of the most prolific histamine former, Morganella morganii, was developed.16S rDNA targeted PCR primers were designed, and the primer specificity and sensitivity of the PCR assay were evaluated. The 16S rDNA sequence (1,503 bp) for M. morganiishowed 95% identity to those for enteric bacteria, i.e., Enterobacterspp., Klebsiellaspp., Citrobacterspp., Hafnia alvei, Proteusspp., and Providenciaspp. The unique primers for M. morganiiwere designed on the basis of the variable regions in the 16S rDNA sequence. The primers showed positive reactions with all M. morganiistrains tested. However, PCR amplification was not detected when the primers were tested with other enteric or marine bacteria. When the sensitivity of the assay was evaluated, M. morganiiwas detected at levels ranging from 106to 108CFU/ml in albacore homogenate after the PCR amplification. The sensitivity of the assay was greatly improved with the enrichment of samples, and 9 CFU of M. morganiiper ml of albacore homogenate was detected after 6 h of enrichment at 37°C.

Ordered differential display PCR was used to identify differentially expressed genes in rat dorsal root ganglia at 7 days following chronic constriction injury (CCI) of the sciatic nerve. Fourteen differentially displayed cDNA bands were isolated, cloned and verified by RT-PCR. The four mRNAs were increased, which included mRNAs encoding heat shock protein 27, fatty acid binding protein, apolipoprotein D and one novel gene. Six down-regulated clones were microtubule-associated protein 1B, protein tyrosine phosphatase α, Kv1.2 channel, myelin protein SR13, medium-sized neurofilament protein, and one novel gene. Our results show that many differentially regulated genes after CCI may play a role in nerve degeneration and/or regeneration and provide a molecular framework for understanding the peripheral mechanism underlying neuropathic pain.

Bacterial growth and histamine formation in Pacific mackerel during storage at 0, 4, 15, and 25°C were monitored. To identify bacterial species contributing to histamine formation, several groups of bacteria were isolated by using selective media under temperatures corresponding to the various storage conditions. Initially, low counts of bacteria were found in the gill, skin, and intestine of fresh fish, and only weak histamine formers were found in the gill. Histamine was found in the muscle when fish were stored above 4°C, and aerobic plate counts reached 106CFU/g. When fish became unsuitable for human consumption by abusive storage, toxicological levels of histamine were always found. The highest level of histamine formed was 283 mg/100 g in 2 days. The optimum temperature for supporting growth of prolific histamine formers was 25°C. The most prolific and prevalent histamine former was Morganella morganii, followed by Proteus vulgaris, both of which were isolated on violet red bile glucose (VRBG) agar. At 15°C, a significant level of histamine was still produced in fish muscle, although prolific histamine formers were less frequently detected than at 25°C. The isolates on thiosulfate citrate bile salts sucrose (TCBS) agar were weak histamine formers and identified as Vibrio parahaemolyticusand Vibrio alginolyticus. At 4°C, less than 57.4 mg/100 g of histamine was found in fish stored for 14 days. Most isolates were natural bacterial flora in the marine environment and identified as weak histamine formers. At 0°C, neither histamine former nor histamine production was detected up to 14 days of storage.

Histamine-producing bacteria were isolated from fresh and temperature-abused albacore using two different isolation procedures. Typically, the bacterial isolates on Niven's or modified Niven's medium produced negligible or low levels of histamine (<300 ppm) in histamine enumeration broth. The most frequently found species using this approach was Hafnia alvei. By prescreening on selective media (eosin methylene blue [EMB] agar for enteric bacteria; deMan Rogosa Sharpe agar for lactic acid bacteria; KF streptococcus agar for streptococci; pseudomonas isolation [PI] agar for pseudomonads; and staphylococcus medium 110 agar for staphylococci) prior to plating on histidine decarboxylase differential media, detection rate of true histamine formers increased. Prolific histamine producers capable of forming >1,000 ppm histamine in culture broth were isolated when PI and EMB agars were used for prescreening. Among the selective media tested, EMB agar was most effective in selecting high histamine producers, as demonstrated by the highest rate of true positives based on histamine analysis. Histamine-producing isolates were mostly enteric bacteria, including Morganella morganii, H. alvei, Klebsiellaspp., Citrobacter freundii, Enterobacterspp., and Serratiaspp. M. morganiiisolated on PI agar from temperature-abused albacore muscle was found to be the highest histamine former. This species was not isolated from fresh albacore, while other enteric bacteria were frequently detected on the gills. However, only a few species isolated from both fresh and temperature-abused muscles were identified as high histamine formers.

Histamine-producing bacteria were isolated from albacore stored at 0, 25, 30, and 37°C. They were screened using Niven's differential medium, and their histamine production was confirmed by high-pressure liquid chromatography analysis. The optimum temperature for growth of histamine-producing bacteria was 25°C. The bacterium producing the highest level of histamine was isolated from fish abused at 25°C. It was identified as Morganella morganiiby morphological, cultural, biochemical, and antimicrobial characteristics and by the Vitek microbial identification system. The M. morganiiisolate was inoculated into tuna fish infusion broth medium, and the effect of temperature was determined for microbial growth and formation of histamine and other biogenic amines. The isolate produced the highest level of histamine, 5,253 ppm, at 25°C in the stationary phase. At 15°C, histamine production was reduced to 2,769 ppm. Neither microbial growth nor histamine formation was detected at 4°C. To determine whether the isolate can also produce other biogenic amines that can potentiate histamine toxicity, production of cadaverine, putrescine, serotonin, tryptamine, tyramine, phenylethylamine, spermidine, and spermine by the isolate was also monitored. Cadaverine, putrescine, and phenylethylamine were detected with microbial growth in the tuna fish infusion broth medium. The optimum temperature for cadaverine, putrescine, and phenylethylamine formation was found to be 25°C, as it was for histamine.

Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc.