Determination of Azide Ions in Blood by Pentafluorobenzyl Derivation Followed by GC-MS / 法医学杂志

Objective To establish a method for determination of the azide ions in blood by gas chromatography-mass spectrometry （GC-MS） following pentafluorobenzyl derivatization. Methods A blood sample of 0.2 mL was placed into a 10 mL glass test tube, and the internal standard sodium cyanide, derivatization reagent pentafluorobenzyl bromide and catalyst tetradecyl benzyl dimethyl ammonium chloride were added in turn. After vortex mixing, the mixture was heated with low-power microwave for 3 min. After centrifugation, the organic phase was taken for GC-MS analysis. Results The azide ions in blood had a good linear relationship in the mass concentration range of 0.5 to 20 μg/mL. The lowest detection limit was 0.25 μg/mL and the relative recovery was 91.36%-94.58%. The method was successfully applied to a case of death from sodium azide poisoning. The mass concentration of azide ions in the blood of the dead was 11.11 μg/mL. Conclusion The method developed in this paper has strong specificity and is easy to operate, which is suitable for the rapid detection of azide ions in blood.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Evaluation of Propidium Monoazide Real-Time PCR for Early Detection of Viable Mycobacterium tuberculosis in Clinical Respiratory Specimens

BACKGROUND: Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification. We evaluated whether PMA real-time PCR is suitable for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respiratory specimens. METHODS: A total of 15 diluted suspensions from 5 clinical MTB isolates were quadruplicated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the DeltaC(T) values (C(T) value in PMA-treated sputum samples-C(T) value in non-PMA-treated sputum samples) between culture-positive and culture-negative specimens. Real-time PCR was performed using Anyplex MTB/NTM Real-Time Detection (Seegene, Korea), and the C(T) value changes after PMA treatment were compared between culture-positive and culture-negative groups. RESULTS: In MTB suspensions, the increase in the C(T) value after PMA treatment was significant in dead cells (P=0.0001) but not in live cells (P=0.1070). In 14 culture-negative sputum samples, the median DeltaC(T) value was 5.3 (95% confidence interval [CI], 4.1-8.2; P<0.0001), whereas that in 69 culture-positive sputum samples was 1.1 (95% CI, 0.7-2.0). In the ROC curve analysis, the cutoff DeltaC(T) value for maximum sensitivity (89.9%) and specificity (85.7%) for differentiating dead from live cells was 3.4. CONCLUSIONS: PMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smear-positive sputum samples.

Binding chemistry and molecular heterogeneity of neurotensin binding protein(s)/receptor in adult chicken tissues.

The study focuses on the importance of Tyr11 amino acid (AA) and subsequent stereochemistry involved in the binding process of neurotensin (NT) with its receptor (NTR)/binding protein(s) as well as the size heterogeneity. Using the binding of 125I-NT with several chicken tissues, it is identified that one of the crucial factors behind all high affinity (Kd ~10 pM) interactions is due to phenolic-OH (Φ-OH) at the para (p) position of Tyr11 within RRPYIL-CO2H (NT8-13) sequence. Replacing the p-OH only in Tyr11 by substituting with p-Cl, p-F and p-NH2 results in significant change of the binding affinity (Kd); p-OH ≈ p-NH2 (~10 pM), p-Cl (~100 pM), p-F (~120 pM). Interestingly, p-NH2 equals to p-OH displaying the highest affinity. Experiments conducted by binding several of the 125I-azido–NT analogs having azido group attached at different positions within the NT molecule have further confirmed the necessity of RRPYIL sequence for high affinity ligand-receptor interaction. The role of Tryp11 in place of Tyr11 in addition to the results above establishes a significant possibility of H–bonding occurring between p-OH of NT and NTR inside the docking space. Photo labeling of the liver tissue by substituted 125I-Y3-azido-NT analogs shows several specifically labeled bands with considerable range of molecular weight (Mr ~90-30 kDa) variations. These results indicate the existence of molecular heterogeneity concerning the sizes of NTR or else any NT binding proteins in the avian tissues. Further, the study has revealed that besides liver, several other chicken tissues also express similar specific high affinity binding (Kd ~20 pM) with varying capacities (Bmax). The order for Bmax is: liver (1.2 pMol/mg) gall bladder (1.03 pMol/mg) > spleen (0.43 pMol/mg) > brain (0.3 pMol/mg) > colon lung (0.15 pMol/mg). In all cases, the binding was reduced by GTPgS (ED50 ~ 0.05 nM), NEM (ED50 ~ 0.50 mM) and NaCl (ED50 ~30 mM), indicating the existence of NTR identical to the mammalian type-1.

A Comparative Study on Mechanical and Biochemical Properties of Bovine Pericardium After Single or Double Crosslinking Treatment

BACKGROUND AND OBJECTIVES: Glutaraldehyde (GA) has been used as a representative method of tissue preservation in cardiovascular surgery. However, GA has showed limited durability including calcification, mechanical failure and toxicity. To overcome those unsolved problems, we analyzed the crosslinking differences of primary amines, GA and genipin in their mechanical and biochemical properties with a single or double crosslinking agent for clinical application. MATERIALS AND METHODS: Samples were divided into 3 groups; control, single crosslinking fixation and double crosslinking fixation after decellurarization using bovine pericardium. For analysis of the biochemical and mechanical properties of each crosslinking method, tensile strength, percentage strain, thermal stability, resistance to pronase, nynhydrin and cytotoxicity test were studied. RESULTS: Combined hexamethylene diamine and suberic acid in the carbodiimide hydrochloride/N-hydroxysucinimide solution (EDC/NHS) after decellurarization, tensile strength and strain percentage were not statistically significant compared to the single crosslinking treated groups (p>0.05). Tissue crosslinking stability was weak in single treatment of diphenylphosphoryl azide, suberic acid, low concentration of EDC, hexamethylene diamine and procyanidin groups, but thermal stability and resistance to the pronase and ninhydrin were markedly increased in concentrated EDC/NHS or after combined double treatment with low concentration of GA or genipin (p<0.001). CONCLUSION: Single or double crosslinking with low concentration of carbodiimide, diphenylphosphonyl azide, procyanidin, suberic acid and hexane diamine were not as effective in mechanical, biochemical, cytotoxic and crosslinking properties compared to GA or genipin fixation, but their mechanical and chemical properties were much improved when combined with low concentrations of GA or genipin in the double crosslinking process.

Assessment of Bronchodilator Responsiveness Following Methacholine-Induced Bronchoconstriction in Children With Asthma

PURPOSE: The aim of this study was to investigate bronchodilator responsiveness (BDR) following methacholine-induced bronchoconstriction and to determine differences in BDR according to clinical parameters in children with asthma. METHODS: The methacholine challenge test was performed in 145 children with mild to moderate asthma, and the provocative concentration causing a 20% decline in FEV1 (PC20) was determined. Immediately after the challenge test, patients were asked to inhale short-acting beta2-agonists (SABAs) to achieve BDR, which was assessed as the change in FEV1% predictedx100/post-methacholine FEV1% predicted. For each subject, the asthma medication, blood eosinophil count, serum total IgE, serum eosinophil cationic protein level, and skin prick test result were assessed. RESULTS: The FEV1 (mean+/-SD) values of the 145 patients were 90.5+/-10.9% predicted, 64.2+/-11.5% predicted, and 86.2+/-11.2% predicted before and after methacholine inhalation, and following the administration of a SABA, respectively. The BDR did not differ significantly according to asthma medication, age, or gender. However, BDR in the atopy group (37.4+/-17.7%) was significantly higher than that in the non-atopy group (30.5+/-10.7%; P=0.037). Patients with blood eosinophilia (38.6+/-18.1%) displayed increased BDR compared with patients without eosinophilia (32.0+/-13.8%; P=0.037). CONCLUSIONS: In children with mild to moderate asthma, the responsiveness to short-acting bronchodilators after methacholine-induced bronchoconstriction was not related to asthma medication, but was higher in children with atopy and/or peripheral blood eosinophilia.

Adventitial Fibroblast Abormality in Thoracic Aortic Aneurysms and Aortic Dissections

BACKGROUND: Development of thoracic aortic aneurysms and aortic dissections (TAAD) is attributed to unbearable wall tension superimposed on defective aortic wall integrity and impaired aortic repair mechanisms. Central to this repair mechanisms are well-balanced and adequately functional cellular components of the aortic wall, including endothelial cells, smooth muscle cells (SMCs), inflammatory cells, and adventitial fibroblasts. Adventitial fibroblasts naturally produce aortic extracellular matrix (ECM), and, when aortic wall is injured, they can be transformed into SMCs, which in turn are involved in aortic remodeling. We postulated the hypothesis that adventitial fibroblasts in patients with TAAD may have defects in ECM production and SMC transformation. MATERIALS AND METHODS: Adventitial fibroblasts were procured from the adventitial layer of fresh aortic tissues of patients with TAAD (Group I) and of multi-organ donors (Group II), and 4-passage cell culture was performed prior to the experiment. To assess ECM production, cells were treated with TNF-alpha (50 pM) and the expression of MMP-2 / MMP-3 was analyzed using western blot technique. To assess SMC transformation capacity, cells were treated with TGF-beta1 and expression of SM alpha-actin, SM-MHC, Ki-67 and SM calponin was evaluated using western blot technique. Fibroblasts were then treated with TGF-beta1 (10 pM) for up to 10 days with TGF-beta1 supplementation every 2 days, and the proportion of transformed SMC in the cell line was measured using immunofluorescence assay for fibroblast surface antigen every 2 days. RESULTS: MMP-3 expression was significantly lower in group I than in group II. TGF-beta1-stimulated adventitial fibroblasts in group I expressed less SM alpha-actin, SM-MHC, and Ki-67 than in group II. SM-calponin expression was not different between the two groups. Presence of fibroblast was observed on immunofluorescence assay after more than 6 days of TGF-beta1 treatment in group I, while most fibroblasts were transformed to SMC within 4 days in group II. CONCLUSION: ECM production and SMC transformation are compromised in adventitial fibroblasts from patients with TAAD. This result suggests that functional restoration of adventitial fibroblasts could well be a novel approach for the prevention and treatment of TAAD.

Molecular Characteristics of Extended Spectrum beta-Lactamases in Escherichia coli and Klebsiella pneumoniae and the Prevalence of qnr in Extended Spectrum beta-Lactamase Isolates in a Tertiary Care Hospital in Korea

PURPOSE: Extended spectrum beta-lactamases (ESBLs) are cephalosporinases that confer resistance to a wide variety of oxyimino cephalosporins and create serious therapeutic problems. In addition, the quinolone resistance qnr genes are becoming increasingly prevalent in clinical isolates, some of which also produce ESBL. This study was designed to evaluate the occurrence and genotypic distribution of ESBL producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) as well as the prevalence and distribution of qnr genes in ESBL-producing isolates in a tertiary care hospital in Korea. MATERIALS AND METHODS: We tested a total of 111 ESBL-producing isolates of E. coli and K. pneumoniae, which were collected at Kyung Hee Medical Center from November 2006 to June 2008. ESBL production was determined by the Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test. The cefotaxime and ceftazidime resistance of the ESBL-producers were transferred to azide-resistant E. coli J53 by conjugation. The presence and identity of ESBL and qnr genes were determined by polymerase chain reaction (PCR) and nucleotide sequencing. RESULTS: The prevalence of ESBLs was 17.7% (297/1,680) of E. coli and 26.5% (240/904) of K. pneumoniae in our hospital during the study periods. Of the 111 collected isolates, 69 isolates were E. coli and 42 isolates were K. pneumoniae. The most prevalent ESBL genotype was CTX-M15. Among the ESBL-producing isolates, 4 E. coli (5.8%) and 17 K. pneumoniae (40.5%) contained qnr genes. qnrB4 was the most frequent type in both E. coli and K. pneumoniae. CONCLUSION: CTX-M15 was the most frequently encountered ESBL. In addition, a high prevalence of qnr genes among ESBL-producing K. pneumoniae was identified in this study.

Selective detection of viable Enterococcus faecalis using propidium monoazide in combination with real-time PCR / 대한치과보존학회지

Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

[Inhibitory effects of NaF-varnish and APF-gel on cariogenic bacteria: an in vitro study]

While there are multiple components of preventive programs developed for caries prevention in children, perhaps none is as important and effective as the appropriate use of fluoride. The primary caries preventive effects of fluoride result from its topical contact with enamel and through its antibacterial actions. Till now bulk of research exists which has compared the antibacterial effects of ordinary topical fluoride gels and solutions. Little or no evidence is seen to tell us which topical fluoride including varnishes is more antibacterial. We suggested further research about antibacterial effect of APF gel and NaF varnish against cariogenic microorganisms [streptococcus mutans and lactobacillus], so use of these may have benefit in reduction of caries. Comparison of inhibitory effect of NaF-varnish versus APF-gel on concentration of cariogenic bacteria [streptococcus mutans and lactobacillus], was the primary goal of this research. In this exprimental study, twenty premolars were sectioned buccolingually. With the use of "window method" certain surfaces of enamel were covered with APF-gel and NaF-varnish. Then, the number of streptococcus mutans and lactobacillus were counted after 18, 24 and 48 hours. In the "Disk diffusion" method the streptococcus mutans with the concentration of 108/ml and volume of 0.1cc were introduced to the M.S.Media culture after application of APF gel and NaF varnish. Then the inhibition zone, measured. Statistical analysis in this research was multilevel modeling. The comparison between gel and varnish after 18, 24 and 48 hours showed that gel has more effect than varnish over the number of lactobacillus. The difference with lactobacillus was statistically significant [p<0.005], but with streptococcus Mutans was not. APF gel was more effective [70.23%] than NaF varnish. Based on the obtained results, APF gel can be used with more thrust than NaF varnish in caries prevention

Grafting and characterization of poly (ethylene glycol) on polysulfone sheets / 生物医学工程学杂志

Grafting of poly (ethylene glycol) (PEG) on the surface of polysulfone (PSF) sheets by simultaneous or sequential UV irradiation with 4-azidobenzoic acid as the photocoupler was carried out. Water contact angle measurements showed that there was a great improvement of hydrophilicity on the grafted surface. X-ray photoelectron spectroscopy suggested that the area covered by PEG be 77.3% and 41.9% respectively after grafting by simultaneous and sequential pathways. With atomic force microscope (AFM), obvious difference in the shape and the phase mode was observed between surfaces of PEG-g-PSF sheets made by these two pathways. Evidences implied that simultaneous pathway would produce a branched PEG layer on the surface, while sequential pathway was coupled with a "pan-cake" PEG layer on it. This study provides the foundation for further advancement in tethering brush-like PEG on PSF hollow fiber membranes.

1-Azido-4-Phenyl-1,4-Butanedione as a convenient precursor for the synthesis of various nitrogen heterocycles

1 -AZIDO-4-phenyl-1,4-butanedione 2 proved to be a convenient precursor for the synthesis of a variety of heterocyclic systems through its treatment with some acidic and basic reagents. For example, 2-benzazepine-1,5-dione 3, 1,3-oxazolane-2,4-dione 4a, 1,3-thiazolane-2,4-dione 4b, 1,3-oxazol-5-one 5, quinazoline-2,4-dione 7, 4,6-diaryl-2-pyrimidineones 9a-d, 2,5-bis-substituted amino-1,3,4-oxadiazole II,5-aryl-2-N-substituted amino-1,3,4-oxadiazoles 13a,b, 1,2,4-triazol-3-ones 14a,b, 1,3-benzoxazine-2,4 [3H]-dione 15 and 1,3,4-oxadiazol-2[3H]-ones 16a,b

Synthesis and reactions of saccharinely-benzoic acid azides. the biological activity of the products

Diaryl ureas are reported to exhibit herbicidal activity and the wide range of pharmaceutical activities of saccharin [1-5] are also well known. In the present investigation we have introduced the saccharinyl moiety into the o -, m-, and p - positions of benzoic acid with the object of studying the behaviour of the resulting acid azides towards base and acid catalyzed decomposition and also to screen the prepared compounds for antimicrobical activities. The o-, m- and p- [saccharinyl] - benzoic acid azides [3a-c] were prepared by reacting o-, m-, and p- saccharinyl benzoic acid chlorides [2a-c] with aq. NaN[3] in acetone

Synthesis and antitumor activity of some hydrazinoacridine hydrazinoacridonanil and 9-oxo-9, 10-dihydroacridin-4-carboxazide derivatives

Six series of acridine derivatives of anticipated antitumor activity have been designed and synthesized. The first comprised 9-[4 sulfamoylphenyl] hydrazinoacridine. The second consisted of 9-[4-sulfamoylphenyl] hydrazinoacridonanil. The third included N- substituted 9-[4 sulfamoylphenyl] hydrazinoacridine-4-yl [N-[4-sulfamoylphenyl]] carboxamide. While the fourth and the fifth included N substituted 9-[4-sulfamoylphenyl]] hydrazinoacridin-4-yl[N- sulfamoylphenyl]] carboxazide derivatives; and the sixth included 9-oxo-9, 10-dihydroacridin-4-yl [N-[4-sulfamoylphenyl]] carboxazide derivatives. The rationale behind the synthesis of these compounds, their methods of synthesis as well as the antitumor activities have been presented

Actividad inhibitoria "in vitro" de algunos antisépticos frente a cultivos de Prototheca wickerhamii / \"In vitro\" inhibitory activity of some antiseptic drugs against Prototheca wickerhamii strains

Se estudió la acción inhibidora "in vitro" de los siguientes antisépticos: etil mercurio tiosalicilato de sodio, azida sódica, borato de sodio, yoduro de potasio, fenol y cloro, frente a cultivos de 5 cepas de Prototheca wickerhamii. La acción "in vitro" de estas drogas fue medida mediante el halo de inhibición que produjeron en los cultivos de Prototheca en medio de agar-miel de Sabouraud incubados a 28ºC. Los resultados obtenidos permitieron establecer que todas las cepas eran uniformemente susceptibles al etilmercurio tiosalicilato de sodio y a la azida sódica, la mayor parte fue inhibida por el yoduro de potasio, en tanto que, el borato de sodio, el fenol y el cloro resultaron inactivos en las diluciones utilizadas

Mutagenicity and anti-mutagenicity of selected spices.

Using Salmonella typhimurium strains TA 100 and TA 1535, the mutagenicity and anti-mutagenicity of extracts of several spices were checked. Spices like pepper, pippali, ginger and mustard increased the number of revertants indicating their mutagenic potential. Garlic extract on the other hand was found to inhibit the mutagenicity produced by direct acting mutagens such as N-methyl N'-nitro-N-nitrosoguanidine and sodium azide. Asafoetida and turmetic extract were found to inhibit microsomal activation dependent mutagenicity of 2-acetamidofluorene. Similar results were also obtained using curcumin and eugenol which are phenolics present in turmeric and clove respectively. These results indicated that some of the spices may ameliorate the effect of environmental mutagens especially present in the food.

Effect of potassium cyanide [KCN] and sodium azide NaN3 on the growth of streptococcus pyogenes Gr A

In order to show that the growth of S. pyogenes Group A was independent of both Fe and O2 as a terminal electron acceptor, KCN and NaN3 were added to the growth medium at levels which completely inhibited growth of aerobic bacteria. These agents have no effect on the growth of S. pyogenes at concentration below 100 mM, proving that the growth was independent of cyt aa3 oxidoreductase and O2