Analysis and application of NAT in Dongguan blood center more than 10 years

This report reviewed the efficacy of nucleic acid testing (NAT), derived from assaying and measuring data, using a Cobass201 system at the Dongguan blood center from 2008 to 2017. During this period, four blood screening models, each reflecting procedure improvements designed to improve residual risk (RR) assessment were assessed. A total of 716 846 blood donors were screened, detecting 1 395 positive by the mixed pool test, which were finally modified to 900 positive cases, after final detection by the separation-single test: detecting 6 HIV cases, 4 HCV cases and 890 HBV cases with a total positive rate of 1.25%. The lowest result was obtained from the twice administered enzyme-linked immunoassay (ELISA) test,used in conjunction with the single Cobas MPX v2.0 model,with rates of: 1/7 405 for HBV, 1/346 020 for HCV and 1/473 934 for HIV, respectively. NAT positive rate is not affected by different screening models. NAT is a recent detection method which can be used to good effect with ELISA, and is a worth while procedure for promoting blood transfusion safety.     

罗氏Cobas c501检测系统尿素分析测量范围的验证及评价

目的:核实并评价罗氏Cobasc501检测系统尿素分析测量范围。方法:主要参照美国临床实验室标准化委员会(NCCLS)指南文件EP6-P的要求,收集含高值待测物的新鲜病人血清,按一定比例混合、离心,计算混合物的浓度并将之作为高值样品(H),与经同样处理获得的低值样品(L)分别按5L、4L+1H、3L+2H、2L+3H、1L+4H、5H的关系配制,形成系列样品,在罗氏Cobasc501检测系统上对各样品的尿素进行检测,每个样品检测4次,数据进行回归分析。结果:回归方程为y=0.9889x+0.19,b=0.9889,介于0.97～1.03之间,ta小于t0.05,说明截距与0无显著性差异,回归直线事实上通过零点。结论:罗氏Cobasc501检测系统检测尿素的分析测量范围为0.7～49.9mmol/L,宽于厂家提供的分析测量范围0.5～40.0mmol/L,完全符合临床检验要求。     

N-acetyltransferase 2 gene polymorphism in patients with colorectal carcinoma

The acetylation polymorphism is a common inherited variation in human drug and carcinogen metabolism. Because N- acetyltransferase (NAT2) is important for the detoxification and/or bioactivation of drugs and carcinogens, polymorphisms of this gene have important implications in therapeutics of and susceptibility to cancer. In this study, NAT2 genotype (NAT2*5A (C(481)T), NAT2*6A (G(590)A), NAT2*7A/B (G(857)A)) and NAT2*14A (G(191)A) and phenotype were determined in 125 patients with colorectal carcinoma and 82 healthy control in Mersin, a city located in the southern region of Turkey. Isolation of the subjects' DNA was performed by using a highly purified PCR template preparation kit/(Roche Diagnostics cat. no: 1 796 828) and the NAT2 polymorphism was detected using real-time PCR (Roche Diagnostics, GmbH, Mannheim, Germany). According to this study high protein intake is associated with the increased risk for the development of colon cancer (OR = 1.73; 95% CI, 1.10-3.07). Although only NAT2*14A fast type was associated with increased risk in patients with colorectal carcinoma (OR = 3.03; 95% CI, 1.56-5.86), when a high protein diet was considered, NAT2*7A/B fast genotype was also found to be associated with an increased risk (OR = 2.06, 95% CI for NAT2*7A/B, 1.10-3.86; OR = 2.65; 95% CI, 1.29-5.46 for NAT2*14A). Smoking status did not differ between the control and patient groups. Our data suggest that exposure to carcinogens through consumption of a high-protein diet may increase the risk of colorectal carcinoma only in genetically-susceptible individuals.     

Comparison of four routinely used vitamin D automated immunoassays

BackgroundTo compare four automated immunoassays for the measurement of 25(OH)-vitamin D (25-OHD) and to assess the impact on the results obtained from a healthy population.MethodsWe analysed 100 serum samples on Unicel DxI 800 (Beckman Coulter), Architect i1000 (Abbott), Cobas e411 (Roche) and Liaison XL (DiaSorin). Passing-Bablok regression and Bland-Altman plots were used for method comparison. In order to categorise the obtained values, results were categorised into the following groups: 0-25 nmol/L, 25-50 nmol/L, 50-75 nmol/L and above 75 nmol/L and compared. The percentage of samples below 75 nmol/L, and below 50 nmol/L was then calculated for every method.ResultsAccording to paired comparisons, each method differs from others (p<0.0001) except Cobas vs Architect, which do not show a statistically significant difference (p=0.39). The strongest correlation was found between Liaison and Architect (ρ=0.94, p<0.0001). The percentage of samples below the recommended value of 75 nmol/L were: 70% (Architect), 92% (Liaison), 71% (Cobas) and 89% (Unicel). The percentage of samples below the value of 50 nmol/L were: 17% (Architect), 55% (Liaison), 28% (Cobas) and 47% (Unicel).ConclusionsThe observed differences stem from the use of different analytical systems for 25-OHD concentration analysis and can result in different outcomes. The recommended values should be established for each assay in accordance with the data provided by the manufacturer or in the laboratory, in accordance with proper standardisation.     

Serotonin N-acetyltransferase (NAT) activity in hen retina and pineal gland: pharmacological induction at noon and antagonism of the light-evoked suppression at night

effective pharmacological procedures are described which markedly increase activity of serotonin N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, during the daytime (in light) and counteract suppressive effects of light on NAI activity at night in the hen retina and pineal gland. Of the tested compounds, and their combinations, the most effective were: “aminophylline + spiroperidol + alpha-methyl-p-tyrosine” for the retina, and “aminophylline + yohimbine (+ alpha-methyl-p-tyrosine)” for the pineal gland. The results give strong support to the concept that the dopaminergic (C₂-receptor) and noradrenergic (alpha₂-adrenergic receptor) mechanisms control NAT activity, and melatonin synthesis, in the hen retina and pineal gland, respectively.     

Experiences in HCV-NAT screening prior to releasing cellular components by the German Red Cross Blood Transfusion Service of Baden-Württemberg.

In this report we present the accumulated data on nucleic acid testing (NAT) for hepatitis C virus (HCV) RNA of blood donations by the Blood Transfusion Service of Baden-Württemberg in the period between March 1997 and March 1999. An extra barcoded blood sample was collected from each donor. Samples were tested by NAT in mini-pools of maximally 96 samples. First-time and repeat donors were tested separately. RT/HCV-PCR was performed with the COBAS HCV Amplicortrade mark, versions 1.0 and 2.0 from Roche Diagnostic Systems.Many modifications have been introduced to the original protocol since the implementation of NAT screening aiming at an increase in the sensitivity and specificity of the assay. NAT positive pools containing serologically positive samples were detected. Initially, reactive pools were identified that could not be confirmed by secondary pooling and single testing procedures. So far, no serologically negative but NAT positive sample has been found.     

Pyridoxal 5′-phosphate related changes in retention of 1,25-dihydroxy vitamin D-receptor ligands in rat intestinal mucosa cell nuclei

After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5′-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5′-phosphate pools in the nuclei: a “mobile” pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5′-phosphate), and a “stable” pool, which was independent of cytoplasmic fluctuations of pyridoxal 5′-phosphate (about 9 pmol pyridoxal 5′-phosphate/mg DNA). Reduction in pyridoxal 5′-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 ± 19.7 vs 762 ± 291 fmol/mg DNA, mean ± SEM). The degree of 1,25-dihydrovitamin D accumulation in the nuclei appeared to be an exponential function of the “mobile” nuclear pyridoxal 5′-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5′-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r=−0.95). These data suggest that pyridoxal 5′-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5′-phosphate with the vitamin D receptor protein in the nuclei.     

印度块菌-云南松菌根际土壤细菌的种群组成和群落结构

以印度块菌-云南松菌根际土壤细菌为研究对象,研究其种群组成和结构特征。(1)稀释平板法分离得到印度块菌-云南松菌根际土壤细菌的纯培养菌株,对菌株的16S rRNA序列测序分析,对测序的菌株数量和得到的OTUs数量绘制物种累积曲线,当物种累积曲线趋于平缓时,对OTUs进行系统发育分析,揭示可培养细菌的种群组成和结构特征。(2)对印度块菌-云南松菌根际土壤细菌16S rRNA基因的V3—V4区进行高通量测序,分析全部细菌类群的种群组成和结构特征。(1)分离得到菌根际可培养细菌793株,分属于3个属的61个OTUs,其中假单胞菌属(Pseudomonas)序列占总序列的86%,不动杆菌属(Acinetobacter)序列占总序列的9.8%,链霉菌属(Streptomyces)序列占总序列的6.5%。假单胞菌是印度块菌-云南松菌根际土壤可培养细菌的绝对优势类群。(2)高通量测序得到菌根际细菌序列8937条,分属于20个门、198属、2073个OTUs。隶属于变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和酸杆菌门(Acidobacteria)的OTUs占总OTUs的65.9%,变形菌门、放线菌门和酸杆菌门细菌是印度块菌-云南松菌根际土壤细菌的优势细菌。隶属于黄杆菌属(Flavobacterium)、根瘤菌属(Rhizobium)和假黄色单胞菌属(Pseudoxanthomona)的OTUs占总OTUs的33%,黄杆菌属、根瘤菌属和假黄色单胞菌属细菌是印度块菌-云南松菌根际土壤细菌的优势属。印度块菌-云南松菌根际土壤可培养细菌多样性较低,假单胞菌属细菌占据绝对优势地位。印度块菌-云南松菌根际土壤细菌类群具有较高的多样性,物种种类丰富,优势菌群集中。     

Mode analysis of a tubular structure of coupled non-linear oscillators for digestive-tract modelling

A commonly accepted mathematical model for the slow-wave electrical activity of the gastro-intestinal tract of humans and animals comprises a set of interconnected nonlinear oscillators. Using a van der Pol oscillator with third-power conductance characteristics as the unit oscillator a number of structures have been analysed using a matrix Krylov-Bogolioubov method linearisation. The mode analysis of one-dimensional chains and two-dimensional arrays has been reported. In this paper the method has been extended to consider a tubular structure which is relevant to modelling small-intestinal rhythms. It is shown that this structure is capable of producing stable single models, non-resonant double modes and degenerated modes. General expressions are obtained for anm×n structure and examples given of two special conditions of 3×4 (i.e. odd numbers of oscillators in a ring) and 4×3 cases. The analytical results obtained for these two cases have been vertified experimentally using an electronic implementation of coupled van der Pol oscillators. Results obtained using fifth-power non-linear oscillators are summarised.     

Ensouled bodies: The continuity of the theme of ‘the body as instrument’

This paper deals squarely with the concept of instrument, specifically, a music instrument, as a unifying agent. The topic occupied writers from Plato, through Plotinus (3rd century, CE), writers within the period known as the European Latin-based Middle Ages, and, as we will see, beyond. Seen from a historical perspective as it recurs, the topic of “body as instrument” is evidence that mind–body interactive reciprocity is not by any means solely a recent multifaceted biochemical, neurological, psychological, and cognitive, subject of inquiry; but, rather, constituted a carefully-articulated topic of discussion in the past – and, that the results of this encounter of various points of view are indeed useful for us today. Further, that alternative modes of knowledge transmission exist, for example, the allegorical mode.     

Liposome encapsulation of the internal control for whole process quality assurance of nucleic acid amplification-based assays

A system intended for whole process quality assurance of nucleic acid amplification assays was developed based on the use of liposomes as cell-mimicking vehicles for the internal control, allowing introduction of the internal control directly into the crude biological specimens. By the proof of principle testing, the Roche Cobas Amplicor CT assay was chosen as model system and the Roche CT/NG Internal Control was thus loaded into the liposomes. The liposome/DNA particles were spiked into a Chlamydia trachomatis-positive urine specimen. A quantitative "in-house" duplex real-time C. trachomatis PCR assay showed that liposomes having Blue Dextran 2000 polysaccharide co-entrapped were the most suited particles as they were efficiently deposited by the centrifugation carried out according to the Roche urine specimen preparation procedure. Furthermore, it was demonstrated that the liposome/DNA particles might be used for whole process quality assurance of Amplicor assay without major modifications of the assay protocol. An additional feature of the use of these liposomes was that the pellet became blue coloured and that might facilitate a thorough removal of the urine supernatant without increasing the risk of disturbance of the pellet. Principally, the liposome/internal control system is versatile and seems to be applicable for whole process quality control of amplification-based assays for detection of various pathogens.     

Effect of a Pool of Peptides Isolated from <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis> (South American Rattlesnake) Venom on Glucose Levels of Mice Fed on a High-Fat Diet

This study investigated the effect of a pool of peptides, isolated from venom of Crotalus durissus terrificus (South American rattlesnake) on glucose concentration in C57BL/6 mice fed on a high-fat diet for 6 weeks. The pool of peptides (molecular mass around of 10 kDa) was obtained using a MidJet apparatus with a cartridge of 10 KDa. The peptide pool was injected intraperitoneally in mice in a single dose (0.5 mg/animal) or multiple doses (0.2 mg/dose). After predetermined times (30, 60, 90 and 120 min) post injections, venous blood samples were collected for enzymatic measurement of serum glucose using a commercial glucose kit (glucose oxidase method). High-fat fed mice showed an increase in blood glucose concentration, in comparison with mice fed on the chow diet. Thirty minutes after a single dose of the peptide pool, high-fat fed animals showed a significant decrease (~47%) in glycemia. However, the glucose level increased again at 60 and 120 min. Conversely, after multiple injections of the pool of peptides administered every 30 min, the blood glucose concentration in the high-fat mice was significantly decreased (~37%) and remained at low levels until 120 min. These results suggest that the tested pool of peptides from Crotalus durissus terrificus contained a peptide (or peptides) with a beneficial role on glucose-lowering action of high-fat fed mice.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly, endogenous coenzymeA was present in the active site. The structure suggests that, contrary to the other prokaryotic NATs, (BACAN)NAT1 possesses a 14-residue insertion equivalent to the “mammalian insertion”, a structural feature considered unique to mammalian NATs. Moreover, (BACAN)NAT1 structure shows marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial NATs.     

Multiway analysis applied to time‐resolved chemiluminescence for simultaneous determination of paracetamol and codeine in pharmaceuticals

This method is based on the enhancing effect of codeine (COD) and paracetamol (PAR) on the chemiluminescence (CL) reaction of Ru(phen)₃²⁺ with Ce(IV). In the batch mode, COD gives a relatively sharp peak with the highest CL intensity at 4.0 s, whereas the maximum CL intensity of the PAR appears at ~60 s after injection of Ce(IV) solution. Whole CL time profiles allowed use of the time‐resolved CL data in combination with multiway calibration techniques, as multiway partial least squares (N‐PLS), for the quantitative determination of both COD and PAR in binary mixtures. In this work, we found that the impact of Ce(IV) concentration on the CL intensity was different for COD and PAR. Therefore, a Ce(IV) concentration mode was added to the time and sample modes to obtain 3D data. The percent relative standard deviation (%RSD) values for 10 determinations of 1.0 × 10^?5 mol/L of COD and 1.0 × 10^?4 mol/L of PAR were 6.1% and 8.7%, respectively. The limit of detection (LOD) values (S/N = 3) were 0.9 × 10^?8 mol/L and 1.0 × 10^?6 mol/L for COD and PAR, respectively. The proposed method was successfully applied to the determination of PAR and COD in commercial pharmaceutical formulations. Acceptable recoveries (90–110%) were obtained for the quantification of these drugs in the real samples. Copyright © 2016 John Wiley & Sons, Ltd.     

N-acetyltransferase 2 polymorphism in patients with diabetes mellitus

The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. Human arylamine NATs are known to exist as two isoenzymes, NAT1 and NAT2. The objective of this study was to identify whether the genetic polymorphism of NAT2 plays a role in susceptibility to Diabetes Mellitus (DM). Ninety-seven patients with DM and 104 healthy controls were enrolled in the study. NAT2*5A, NAT2*6A, NAT2*7A/B and NAT2*14A polymorphisms were detected by using real time PCR with LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). According to our data, the NAT2*5A and NAT2*6A mutant genotypes and NAT2*14A heterozygous genotype were associated with an increased risk of development of DM (OR = 47.06; 95%CI: 10.55-209.77 for NAT 2*5A, OR = 18.48; 95%CI: 3.83-89.11 for NAT2*6A and OR = 18.22; 95%CI: 6.29-52.76 for NAT2*14A). However, the NAT2*7A/B gene polymorphism carried no increased risk for developing DM disease. After grouping according to phenotypes as either slow or fast acetylators, NAT2*6A slow acetylator was found to be a significant risk factor for DM (OR = 6.09; 95%CI: 1.99-18.6, p = 0.02). The results indicate that NAT2 slow acetylator genotypes may be an important genetic determinant for DM in the Turkish population.     

Automatic control for fed-batch culture of single cell protein

Candida utilis was cultivated in a 5-liter jar fermentor using ethanol as sole carbon source. Control of ethanol in the cultivation broth was performed by using an ethanol vapor monitoring instrument and an oxygen electrode coupled with two control circuits. By setting upper and lower bounds according to the predetermined conditions, a signal from a gas monitoring sensor switched the lower or higher bound relay governing the actuating or switch-off of the motor; this maintained a proper concentration of ethanol in the cultivation of ethanol in the system. The growth of cells was found to be satisfactory. Cell concentration reached 64 g/liter during a 20-hr cultivation. As the results of comparative experiments, the control mode using the gas monitoring instrument was found to be superior to that using dissolved oxygen as a controlling signal, especially at high cell concentration.     

Limit of blank, limit of detection and limit of quantitation

* Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure. * LoB is the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested. LoB = mean(blank) + 1.645(SD(blank)). * LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. LoD is determined by utilising both the measured LoB and test replicates of a sample known to contain a low concentration of analyte. * LoD = LoB + 1.645(SD (low concentration sample)). * LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. The LoQ may be equivalent to the LoD or it could be at a much higher concentration.