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INTRODUCTION: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. METHODS: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. RESULTS: Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p

Batch experiments with Na2S as the sulfur were used to investigate the effects of different S/N（molar ratios） on the denitrification performance of sulfide autotrophic denitrification and nitrite accumulation.The results showed that the removal effect of NO-3-N and NO-X-N increased significantly with the increase of S/N when it ≥ 2.80,the removal rate of NO-3-N and NO-X-N increased by 3.7 times and 3.6 times in the first 6 h respectively when S/N increased from 0.35 to 2.80,but the removal effect of NO-3-N and NO-X-N changed little when it increased from 2.80 to 4.20.Stable NO-2-N accumulation can be obtained in the system when S/N≤0.70,and the NO-2-N accumulation rate（0.35,0.70） can reach 47.9% and 24.5% at the end of the reaction. However, it is difficult to stably accumulate NO-2-N when S/N≥1.40,and the highest accumulation rate（1.40,2.80,4.20） of NO-2-N was 55.0%,33.3% and 39.7%.Stable NO-2-N accumulation can be obtained when S/N≤0.70,while increasing S/N will provide more electrons for denitrification, alleviate the competition of various denitrifying enzymes for electrons, reduce the accumulation of intermediates, and improve the effect of denitrification when it is in the range of 1.40～2.80. [ABSTRACT FROM AUTHOR]

Advances in Raman instrumentation have led to the implementation of a remote dispersive Raman spectrometer on the Perseverance rover on Mars, which is used for remote sensing. For remote applications, dispersive spectrometers suffer from a few setbacks such as relatively larger sizes, low light throughput, limited spectral ranges, relatively low resolutions for small devices, and high sensitivity to misalignment. A spatial heterodyne Raman spectrometer (SHRS), which is a fixed grating interferometer, helps overcome some of these problems. Most SHRS devices that have been described use two fixed diffraction gratings, but a variance of the SHRS called the one-grating SHRS (1g-SHRS) replaces one of the gratings with a mirror, which makes it more compact. In a recent paper we described monolithic two-gratings SHRS, and in this paper, we investigate a single-grating monolithic SHRS (1g-mSHRS), which combines the 1g-SHRS with a monolithic setup previously tested at the University of South Carolina. This setup integrates the beamsplitter, grating, and mirror into a single monolithic device. This reduces the number of adjustable components, allows for easier alignment, and reduces the footprint of the device (35 × 35 × 25 mm with a weight of 80 g). This instrument provides a high spectral resolution (∼9 cm−1) and large spectral range (7327 cm−1) while decreasing the sensitivity to alignment with a field of view of 5.61 mm at 3m. We discuss the characteristics of the 1g-mSHRS by measuring the time-resolved remote Raman spectra of a few inorganic salts, organics, and minerals at 3 m. The 1g-mSHRS makes a good candidate for planetary exploration because of its large spectral range, greater sensitivity, competitively higher spectral resolution, low alignment sensitivity, and high light throughput in a compact easily aligned system with no moving parts. Graphical Abstract This is a visual representation of the abstract. [ABSTRACT FROM AUTHOR]

In this study, three welding methods are used. The purpose to investigation the effects of SMAW, SAW, and gas tungsten arc welding (GTAW) on the tensile stress of low carbon steel conforming to ASTM 283 c. 8mm thick plates are used as base material for butt welded joints. The tensile properties of the welded joints were evaluated and the results were compared by experts using the Taguchi method to design three levels of each parameter (current, voltage and displacement speed). From this research, it is found that compared to metal shielded arc welding and submerged arc welding, the pulling effect of the gas shielded welding joint of the tungsten electrode is the best. This is mainly due to the presence of The results of using analysis of variance (ANOVA) to estimate important parameters show that welding current and speed of the weld have a significant effect on tensile stress .the experimental results are in agreement with predicted results, and the maximum error is 3%..

In this paper the fracture toughness of WC-12Co coatings in optimum particle temperature in high velocity oxy fuel (HVOF) process have been studied by means of Vickers indentation. Multiple linear regression model applying Minitab, were used to determine the relationship and interaction between HVOF parameters and particle temperature. For genetic algorithm optimization, the signal to noise ratio was applied as a functional output of design of experiments. The results of validation test show a good agreement between obtained optimum condition and the results of genetic algorithm. The fracture toughness obtained by Vickers indentation shows the direct effect of particle temperature on coating toughness. The maximum amount of signal-to-noise using the genetic algorithm for velocity and temperature is 53.07 and -64.62, which equals 450.2 m/s and 1702 ºC respectively. The results show that the Fracture toughness of WC-12Co deposited by LPG fuel in smallest level of temperature is 2.83MPa(m)1/2 compared to 1.32MPa(m)1/2 in highest temperature. The spray watch diagnostic system, micro-hardness test, Vickers indentation, X-Ray diffraction, EDS and scanning electron microscopy have been used for this purpose.

Sum frequency generation (SFG) * Equal contributors. spectroscopy was used to deduce the orientation of the terminal methyl (CH3) group of self-assembled monolayers (SAMs) at the air–solid and air–liquid interfaces at surface concentrations as low as 1% protonated molecules in the presence of 99% deuterated molecules. The SFG spectra of octadecanethiol (ODT) and deuterated octadecanethiol (d37 ODT) SAMs on gold were used for analysis at the air–solid interface. However, the eicosanoic acid (EA) and deuterated EA (d39 EA) SAMs on the water were analyzed at the air–liquid interface. The tilt angle of the terminal CH3 group was estimated to be ∼39 ° for a SAM of 1% ODT : 99% d37 ODT, whereas the tilt angle of the terminal CH3 group of the 1% EA : 99% d39 EA monolayer was estimated to be ∼32 °. The reliability of the orientational analysis at low concentrations was validated by testing the sensitivity of the SFG spectroscopy. A signal-to-noise (S/N) ratio of ∼60 and ∼45 was obtained for the CH3 symmetric stretch (SS) of 1% ODT : 99% d37 ODT and 1% EA : 99% d39 EA, respectively. The estimated increase in S/N ratio values, as a measure of the sensitivity of the SFG spectroscopy, verified the capacity to acquire the SFG spectra at low concentrations of interfacial molecules under ambient conditions. Overall, the orientational analysis of CH3 SS vibrational mode was feasible at low concentrations of protonated molecules due to increased S/N ratio. In support, the improved S/N ratio on varying incident power density of the visible beam was also experimentally demonstrated. [ABSTRACT FROM AUTHOR]

Assuming that a time series incorporates "signal" and "noise" components, we propose a method to estimate the extent of the "noise" component by considering the smoothing properties of the state-space of the time series. A mild degree of smoothing in the state-space, applied using a Kalman filter, allows for noise estimation arising from the measurement process. It is particularly suited in the context of a reputation index, because small amounts of noise can easily mask more significant effects. Adjusting the state-space noise measurement parameter leads to a limiting smoothing situation, from which the extent of noise can be estimated. The results indicate that noise constitutes approximately 10% of the raw signal: approximately 40 decibels. A comparison with low pass filter methods (Butterworth in particular) is made, although low pass filters are more suitable for assessing total signal noise. [ABSTRACT FROM AUTHOR]

In this paper the fracture toughness of WC-12Co coatings in optimum particle temperature in high velocity oxy fuel (HVOF) process have been studied by means of Vickers indentation. Multiple linear regression model applying Minitab, were used to determine the relationship and interaction between HVOF parameters and particle temperature. For genetic algorithm optimization, the signal to noise ratio was applied as a functional output of design of experiments. The results of validation test show a good agreement between obtained optimum condition and the results of genetic algorithm. The fracture toughness obtained by Vickers indentation shows the direct effect of particle temperature on coating toughness. The maximum amount of signal-to-noise using the genetic algorithm for velocity and temperature is 53.07 and -64.62, which equals 450.2 m/s and 1702° C respectively. The results show that the Fracture toughness of WC-12Co deposited by LPG fuel in smallest level of temperature is MPa(m)1/2 compared to MPa(m)1/2 in highest temperature. The spray watch diagnostic system, microhardness test, Vickers indentation, X-Ray diffraction, EDS and scanning electron microscopy have been used for this purpose. [ABSTRACT FROM AUTHOR]

Assuming that a time series incorporates “signal” and “noise” components, we propose a method to estimate the extent of the “noise” component by considering the smoothing properties of the state-space of the time series. A mild degree of smoothing in the state-space, applied using a Kalman filter, allows for noise estimation arising from the measurement process. It is particularly suited in the context of a reputation index, because small amounts of noise can easily mask more significant effects. Adjusting the state-space noise measurement parameter leads to a limiting smoothing situation, from which the extent of noise can be estimated. The results indicate that noise constitutes approximately 10% of the raw signal: approximately 40 decibels. A comparison with low pass filter methods (Butterworth in particular) is made, although low pass filters are more suitable for assessing total signal noise.

Limiting the noise exposure of people to limit the risk of hearing damage is the preliminary aim of hearing conservation programs and regulations of most of the countries. Along with the performance and aesthetic considerations; noise reduction carries equal importance in product design and development as manufacturers are obligated for quieter product due to stringent legislations. The most restrictive factor for severity assessment of noise is the cost. This study describes a low cost acoustic test chamber having free working space of 2.28m x 2.28m x 1.12m (7.48ft x 7.48ft x 3.68ft) designed with multilayer absorption treatment by giving emphasis on Green Technology (Green Tech) for sound pressure level measurements of small size sources. The chamber performance has been assured by different methods of validation viz. S/N, 1 Watt, 1 kHz, 1m and Inverse square law (1/r² or 6 dB/dd). Under realistic conditions of ambient noise the A-weighted background noise level is found to be 21 dB which is low enough to make the use of acoustic test chamber for designed purposes. Researchers can adopt these methodologies with the use of common laboratory equipments to validate the acoustic chamber. This study attempts to bridge the gap between optimal acoustic conditions and cost of acoustic chamber. [ABSTRACT FROM AUTHOR]

The influences of column hardware, such as chromatographic tubes and frits, on liquid chromatography–mass spectrometry (LC–MS) analysis of phosphorylated compounds were evaluated. The signal to noise ratio (S/N) and the intensity of flavin adenine dinucleotide (FAD) using a glass lined tube and polyethylene frit (GL–PE) column was approximately 170 and 90 times higher, respectively, than those using conventional stainless steel tube and stainless steel frit (S–S) column. In addition, the retention time of FAD using GL–PE column was the shortest compared to other columns. Interaction between phosphorylated compounds and metal ions in the flow path in the S–S column was stronger than that between them and the GL–PE column. Thus, the metal ions in the flow path in GL–PE column were low. Since the specific surface area of a pair of frits was 70 times larger than that of a chromatographic tube (150 mm × 2.1 mm), the frits were found to have more effective improvement of the S/N as well as the intensity than the chromatographic tubes, when phosphorylated compounds were analyzed by LC–MS. When the evaluated phosphorylated compounds were analyzed by LC–MS(/MS) using a GL–PE column, the intensity and S/N were increased. [ABSTRACT FROM AUTHOR]

Present investigation deals with the effect of sulfide to nitrate (S/N) molar ratio on the simultaneous anaerobic sulfide and nitrate removal on capacity, stability and selectivity of the process. The volumetric sulfide-sulfur and nitrate-nitrogen removal rates at molar S/N ratio of 5:2 were 4.86kg(m3 d)−1 and 0.99kg(m3 d)−1, respectively, which were higher than those at S/N molar ratios of 5:5 and 5:8. Moreover, the fluctuations in the effluent at S/N ratio of 5:2 were less than those at the other two tested ratios. During the operation, the ratio of converted sulfide to converted nitrate tended to approach 5:2. The selectivity for elemental sulfur and dinitrogen was improved when the S/N molar ratio was set at 5:2 rather than 5:5 or 5:8. The process became unstable if the influent sulfide surpassed its critical concentration. The electron balance between reactants was also analyzed for different S/N molar ratios. [Copyright &y& Elsevier]

Abstract: Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future. [Copyright &y& Elsevier]

Background: Titer methods are commonly used to characterize the magnitude of an antidrug antibody response. Assay S/N is an appealing alternative, but the circumstances under which use of signal-to-noise (S/N) is appropriate have not been well defined., Results: We validated both titer and S/N-based methods for several therapeutics. S/N correlated strongly with titer both in aggregate and when examined on a per subject basis. Analysis of impact of antibody magnitude on pharmacokinetics yielded the same result using either method. Each assay demonstrated excellent precision, good linearity, and adequate drug tolerance., Conclusion: Under these circumstances, assay S/N is a valid alternative to titer for assessment of the magnitude of an antidrug antibody response.

Methylmalonic aciduria (MMA) is one of the most frequent organic acidurias, a class of diseases caused by enzymatic defects mainly involved in the catabolism of branched-chain amino acids. Recently, mild MMA and C4-dicarboxylyl-carnitine (C4DC-C) accumulation have been reported in patients carrying mutation in genes encoding the α-subunit (SUCLG1) and the β-subunit (SUCLA2) of the ADP-forming succinyl-CoA synthetase (SCS). We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify in dried blood spot the two isobaric compounds of C4DC-C, succinyl-carnitine and methylmalonyl-carnitine, to allow the differential diagnosis between classical MMA and SCS-related defects. This method, with an easy liquid-phase extraction and derivatization procedure, has been validated to demonstrate the specificity, linearity, recovery, lowest limit of quantification (LLOQ), accuracy and precision for quantitative determination of blood succinyl-carnitine and methylmalonyl-carnitine. The assay was linear over a concentration range of 0.025-10 μmol/L and achieved the LLOQ of 0.025 μmol/L for both metabolites. The average slope, intercept, and coefficient of linear regression (r(2)) were respectively: 0.3389 (95% confidence interval 0.2888-0.3889), 0.0113 (95% confidence interval -0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990-1.0000) for succinyl-carnitine and 0.5699 (95% confidence interval 0.5263-0.6134), 0.0319 (95% confidence interval -0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995-1.0000) for methylmalonyl-carnitine. Within-day and between-day coefficients of variation (CV) were 1.94% and 3.19% for succinyl-carnitine and 3.21%, and 2.56 for methylmalonyl-carnitine. This method is accurate and provides a new tool to differentiate patients with classical methylmalonic acidemia from those with SCS-related defects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A total of 27 shallow groundwater samples were collected from the Taihu Lake region (TLR), to determine the concentrations of 14 organochlorine pesticide (OCP) species, identify their possible sources, and estimate health risk of drinking the shallow groundwater. All OCP species occurred in the shallow groundwater of TLR with high detection frequency except p, p'-dichlorodiphenyldichlorothane (p, p'-DDD) and p, p'-dichlorodiphenyltrichloroethane (p, p'-DDT). DDTs and hexachlorocyclohexanes (HCHs) were the dominant OCP contaminants in the shallow groundwater of TLR, and they account for 44.2% total OCPs. The low α-HCH/γ-HCH ratio, high β-HCH/(α+γ)-HCH ratio and β-HCH being the dominant HCH isomers for the majority of samples suggest that the HCHs were mainly from the historical use of lindane after a period of degradation. p, p'-DDE being the dominant DDT metabolite for all the samples indicated that the DDTs were mainly from the historical residues. Compositional analysis also suggested that there were fresh input sources of heptachlors, aldrins and endrins in addition to the historical residues. Correlation analysis indicated the hexachlorobenzene (HCB) impurity in the shallow groundwater of TLR was likely from the historical application of lindane and technical HCH (a mixture of HCH isomers that is produced by photochlorination of benzene). Carcinogenic risk values for α-HCH, heptachlor, heptachlor epoxide, aldrins and dieldrin in the shallow groundwater in majority area of TLR were found to be >10(-6), posing a potentially serious cancer risk to those dependant on shallow groundwater for drinking water., (© 2013.)

An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50min on a Mac-Mod ACE 3 C18 column (150mm×4.6mm i.d., 3μm) with a gradient mobile phase comprising 20-95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC-quadrupole TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1-0.3mg/mL, r≥0.9998), accuracy (recovery 99.7-101.1%), precision (intra-lab RSD≤1.39%, inter-lab RSD≤0.91%), sensitivity (LOD 0.08μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Ganciclovir/valganciclovir plays an important role in the treatment and prevention of cytomegalovirus disease after organ transplantation., Material and Methods: We developed and validated a simple chromatographic method by ultra-performance liquid chromatography tandem mass spectrometry to measure plasma concentration of ganciclovir in human plasma. Chromatographic separation was achieved using an Acquity(®) UPLC(®) BEH™ (2.1×50mm id, 1.7μm) reverse-phase C18 column, with a water/methanol linear gradient containing ammonium acetate/formic acid at a 0.4mL/min flow rate. Ganciclovir and its internal standard (acyclovir) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode., Results: The limit of detection and quantification were 0.03 and 0.06mg/L, respectively, and linearity was observed between 0.06 and 30.0mg/L. Intra-day and day-to-day coefficients of variation and relative biases ranged from 3.6 to 5.4%, 4.2 to 6.2%, -2.6 to -1.1% and -4.0 to -2.8%, respectively. Recovery values were greater than 81.9%. Evaluation of the matrix effect showed ion suppression for ganciclovir and acyclovir. No carry-over was observed., Conclusions: The validated method is useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to the daily clinical laboratory practice to measure the concentration of ganciclovir in human plasma., (© 2013.)

The artificial sweeteners sucralose (SCL), acesulfame (ACS), saccharin (SAC), and cyclamate (CYC) have been detected in environmental waters in Europe and North America. Higher environmental levels are expected in view of the increasing consumption of these food additives. In this study, an isotope-dilution mass spectrometry (IDMS) LC-MS/MS method was developed and validated for quantifying the four artificial sweeteners in boreal lakes (n = 3) and rivers (n = 12). The highest concentrations of ACS, SAC, CYC and SCL were 9,600, 490, 210 and 1000 ng/L, respectively. ACS and SAC were detected in all studied samples, and CYC and SCL in 98% and 56% of the samples. Seasonal trends of ACS and SAC were observed in some rivers. ACS and SCL concentrations in rivers correlated linearly with population equivalents of the wastewater treatment plants in the catchment areas, whereas SAC and CYC concentrations depend more on the source., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Hepatitis E virus (HEV) genotype 3 is recognised as an emerging pathogen in industrialised countries. The currently commercially available HEV-specific enzyme linked immunosorbent assays (ELISAs) are primarily designed for the detection of antibodies against genotypes 1 (Burma) and 2 (Mexico) and may not sensitively detect HEV genotypes 3 or 4., Objectives: This study aimed to evaluate the analytical and clinical performances of eight commercially available HEV serum antibody immunoglobulin M (IgM)- and immunoglobulin G (IgG)-specific ELISAs for genotype 1 and 3 HEV infections in a clinical setting and to study the antibody responses against HEV of immunocompromised versus immunocompetent patient groups., Study Design: Analytical performance and diagnostic sensitivity and specificity were assessed using well-defined reference samples and samples from patients with polymerase chain reaction (PCR)-confirmed HEV infection (n=88) and a specificity panel (n=98)., Results: Limiting dilutions indicated that the highest analytical sensitivity in head-to-head comparison was measured for the Mikrogen_new IgG assay. Taking the serum working dilutions of each assay into account, the Wantai IgG assay was the most sensitive assay. Receiver operator curve (ROC) analysis showed area under the curve (AUC) values of 0.943, 0.964, 0.969, 0.971, 0.974 and 0.994 for the DSI, Mikrogen_old, MP Diagnostics, Mikrogen_new, Wantai and DiaPro anti-HEV IgM assays, respectively. The highest specificity of currently available assays was found for the IgM Wantai assay (>99%). If anti-HEV IgM and IgG results from each supplier were combined, DSI and Wantai assays were able to detect the highest number of (passed) HEV infections., Conclusions: Our study showed that current commercial HEV ELISAs could be used to diagnose HEV genotype 3 infection adequately in a clinical setting., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: Patients affected by Phenylketonuria (PKU) require lifelong management based on phenylalanine (Phe) and tyrosine (Tyr) restricted intake or tetrahydrobiopterin (BH4) administration. Frequent monitoring of blood concentration of both amino acids during treatment is the key point for clinicians to achieve the best long-term neuropsychological outcome., Results: The present study develops and validates a rapid and simple method for Phe and Tyr quantization in dried blood spot (DBS) since this specimen has the advantage of being low invasive, easily withdrawn even at home and stable if mail-delivered. The validation studies showed the robustness of the method., Conclusions: Serum and DBS samples from PKU patients were analyzed and compared, finding a good correlation of Phe and Tyr concentrations between the two different matrixes., (© 2013.)

Allitinib, also known as AST1306, is a novel irreversible inhibitor of the epidermal growth factor receptors 1 and 2. Allitinib is currently used in clinical trial to treat solid tumors. A previous study showed that allitinib is extensively metabolized in humans. Amide hydrolysis metabolite (M6) and 29,30-dihydrodiol allitinib (M10) are the major metabolites in circulation. To study the pharmacokinetics of allitinib and its two major metabolites in cancer patients, a rapid, sensitive and reliable LC-MS/MS method was developed and validated for the simultaneous determination of allitinib, M6 and M10 in human plasma. After simple protein precipitation, the analytes and the combined internal standards (lapatinib and NB-2, an analog of allitinib) were separated on a Zorbax Eclipase XDB C18 column (50 mm × 4.6 mm, 1.8 μm, Agilent) using a mobile phase of 5 mM ammonium acetate with 0.1% formic acid (phase A) and 50% (v/v) methanol in acetonitrile (phase B) with gradient elution. Mass spectrometric detection was conducted by atmospheric-pressure chemical ionization in positive ion multiple reaction monitoring modes using AB Sciex Triple Quad 6500 system. Linear calibration curves were obtained for the following concentration range: 0.300-200 ng/ml for allitinib; 0.030-20.0 ng/ml for M6; and 0.075-50.0 ng/ml for M10. Intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all of the concentrations. The method was successfully applied to a preliminary clinical pharmacokinetic study following oral administration of allitinib tosylate tablets in cancer patients., (© 2013 Elsevier B.V. All rights reserved.)

A rapid, sensitive, and enantioselective method was developed and validated for determination of ornidazole enantiomers in human plasma by liquid chromatography-tandem mass spectrometry. Ornidazole enantiomers were extracted from 100μl of plasma using ethyl acetate. Baseline chiral separation (Rs=2.0) was obtained within 7.5min on a Chiral-AGP column (150mm×4.0mm, 5μm) using an isocratic mobile phase of 10mM ammonium acetate/acetic acid (100/0.01, v/v). Stable isotopically labeled R-(+)-d5-ornidazole and S-(-)-d5-ornidazole were synthesized as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive electrospray ionization, using the transitions of m/z 220→128 for ornidazole enantiomers, and m/z 225→128 for d5-ornidazole enantiomers. The method was linear in the concentration range of 0.030-10.0μg/ml for each enantiomer. The lower limit of quantification for each enantiomer was 0.030μg/ml. The relative standard deviation values of intra- and inter-day precision were 1.8-6.2% and 1.5-10.2% for R-(+)-ornidazole and S-(-)-ornidazole, respectively. The relative error values of accuracy ranged from -4.5% to 1.2% for R-(+)-ornidazole and from -5.4% to -0.8% for S-(-)-ornidazole. The validated method was successfully applied to a stereoselective pharmacokinetic study of ornidazole after oral administration of 1000mg racemic ornidazole., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this manuscript, a new method based on the use of off-line dispersive solid-phase extraction (dSPE) combined with ultra-high performance liquid chromatography with diode-array detection was developed to determine 11 sulfonamide antibiotics (sulfanilamide, sulfacetamide, sulfadiazine, sulfathiazole, sulfamerazine, sulfadimidin, sulfamethoxypyridazine, sulfadoxine, sulfamethoxazole, sulfisoxazole and sulfadimethoxine) in mineral waters with different mineral content. For this purpose, pristine multi-walled carbon nanotubes (MWCNTs) and magnetic-MWCNTs (m-MWCNTs) were used as sorbents. Magnetic nanoparticles were synthesized by means of a solvothermal process, assembled onto CNTs through an "aggregation wrap" mechanism and characterized by scanning electron microscopy. Parameters affecting the extraction such as volume and pH of the sample, amount of sorbent and type and volume of eluent were optimized. Once optimum extraction conditions (250 mL of water at pH 6.0 and elution with 25 mL of MeOH) were obtained, the extraction efficiency of the different carbon nanomaterials was compared. Results demonstrated the higher extraction capacity of pristine MWCNTs with recoveries between 61 and 110% (except for sulfacetamide which ranged between 40 and 53%) and between 22 and 77% for m-MWCNTs. Limits of detection lower than 32 ng/L were achieved for all of the analyzed samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Early blindness results in occipital cortex neurons responding to a wide range of auditory and tactile stimuli. These changes in tuning properties are accompanied by an extensive reorganization of the occipital cortex that includes alterations in anatomical structure, neurochemical and metabolic pathways. Although it has been established in animal models that neurochemical pathways are heavily affected by early visual deprivation, the effects of blindness on these pathways in humans is still not well characterized. Here, using (1)H magnetic resonance spectroscopy in nine early blind and normally sighted subjects, we find that early blindness is associated with higher levels of creatine, choline and myo-Inositol and indications of lower levels of GABA within the occipital cortex. These results suggest that the cross-modal responses associated with early blindness may, at least in part, be driven by changes within occipital biochemical pathways., (Copyright © 2013. Published by Elsevier Ltd.)

As with other protein therapeutics, trebananib (AMG 386), an investigational peptide Fc-fusion protein ("peptibody") that inhibits angiogenesis by neutralizing the interaction of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) with the Tie2 receptor, has the potential to trigger an immune response in cancer patients treated with the therapeutic. An electrochemiluminescence bridging anti-drug antibody (ADA) assay that was utilized to support early-phase clinical trials in the development of trebananib was found to lack adequate sensitivity and drug tolerance in later-phase clinical studies when higher doses of trebananib were administered. Therefore, we developed a surface plasmon resonance (SPR) immunoassay method utilizing a secondary confirmatory detector antibody (goat anti-human IgG F[ab']2) known to cross-react with human IgG and IgM to better assess the potential impact of immunogenicity on the pharmacokinetics, pharmacodynamics, and toxicity of trebananib. The SPR method was more sensitive than the electrochemiluminescence bridging assay because of signal amplification from the confirmatory binding of the detector antibody; drug tolerance was improved since antibody binding avidity does not affect detection on this platform. Despite the inability of the confirmatory detector antibody to bind angiopoietins in protein-free buffer, false-positive ADA results were generated from patient serum samples containing Ang1 and Ang2 through an apparently specific binding between the angiopoietins and the confirmatory detector antibody, likely mediated by the interaction of the angiopoietins with serum immunoglobulins. Addition to the sample diluent of a human antibody that specifically binds to Ang1 and Ang2 with high affinity resulted in a complete block of angiopoietin interference without affecting ADA detection. This biosensor-based assay provides a reliable method for assessing immunogenicity in phase 3 clinical trials., (© 2013.)

Background: Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely on clinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine., Methods: The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory. Major sulfatide isoforms are normalized to the least variable component of the spectrum, which is the indigenous C18:0 isoform. This procedure does not require the use of specific internal standards and minimizes errors caused by sample preparation and measurement., Results: Urinary sulfatides were analyzed in a set of 21 samples from patients affected by sulfatidosis. The combined abundance of the five most elevated isoforms, C22:0, C22:0-OH, C24:0, C24:1-OH, and C24:0-OH sulfatides, was found to give the greatest distinction between MLD-affected patients and a control group., Conclusions: The method avoids transportation of liquid urine samples and generates stable membrane-bound sulfatide samples that can be stored at ambient temperature. MS/MS sulfatide profiling targeted on the most MLD-representative isoforms is simple with robust results and is suitable for screening., (© 2013.)

Background: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque., Method: In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization., Results: The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5)., Conclusion: This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions., (© 2013. Published by Elsevier B.V. All rights reserved.)

Gas chromatography mass spectrometry (GC/MS)-based fecal metabonomics represents a powerful systems biology approach for elucidating metabolic biomarkers of lower gastrointestinal tract (GIT) diseases. Unlike metabolic profiling of fecal water, the profiling of complete fecal material remains under-explored. Here, a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) method was developed and validated for the global metabonomic profiling of human feces. Fecal and fecal water metabotypes were also profiled and compared. Additionally, the unclear influence of blood in stool on the fecal metabotype was investigated unprecedentedly. Eighty milligram of lyophilized feces was ultrasonicated with 1mL of methanol:water (8:2) for 30min, followed by centrifugation, drying of supernatant, oximation and trimethylsilylation for 45min. Lyophilized feces demonstrated a more comprehensive metabolic coverage than fecal water, based on the number of chromatographic peaks. Principal component analysis (PCA) indicated occult blood (1mgHb/g feces) exerted a negligible effect on the fecal metabotype. Conversely, a unique metabotype related to feces spiked with gross blood (100mgHb/g feces) was revealed (PCA, R(2)X=0.837, Q(2)=0.794), confirming the potential confounding effect of gross GIT bleeding on the fecal metabotype. This pertinent finding highlights the importance of prudent interpretation of fecal metabonomic data, particularly in GIT diseases where bleeding is prevalent., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We developed a sensitive, reliable, and accurate LC/ESI-MS/MS method for measurement of 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine amides in urine. This atypical C24 bile acid has been reported previously to be present in the urine of patients with Niemann-Pick Type C (NPC) disease. In the method, targeted analytes are concentrated at the front edge of a trapping column, Shim-pack MAYI-C8, which permits elimination of contaminating molecules in the urinary matrix. The trapped analytes are then eluted, separated on a YMC-Pack Pro C18, and quantified with MS/MS using selected reaction monitoring. The method could detect (as amount injected) 2pg of nonamidated 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid, 2pg of its glycine-amide, and 0.6pg of its taurine-amide, and is linear up to 300pg. The method was then used to measure the three analytes in the urine of NPC patients (N=2), 3β-hydroxysteroid dehydrogenase deficiency patients (N=2), and healthy volunteers (N=8). Measurable concentrations of all three analytes were present in all subjects. The urinary concentration of the sum of all three analytes was four hundred times greater in the 3month NPC patient and 40times greater in the adult patient than that of healthy volunteers. The markedly elevated urinary concentration of 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides in NPC patients suggests that these compounds may be valuable biomarkers for detection of NPC disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are involved in a plethora of physiological functions in all kinds of tissues. Detailed knowledge about GPCR presence and expression levels in tissues can be very helpful for drug development as the majority of drugs are designed to modulate membrane receptors. Furthermore, it is known that many adverse drug effects result from GPCR interactions. However, very few satisfactory methods are currently available for the detection and quantification of GPCRs. The detection is complicated by their three-dimensional structure, their hydrophobic properties, and their localization in the plasma membrane with 7-trans-membrane domains and small cytosolic and extracellular domains. Due to these properties it is very difficult to generate specific antibodies directed against GPCRs for sandwich immunoassays and Western blot. We therefore designed an immunoaffinity- and mass spectrometry-based approach to analyze GPCR-specific signature peptides in tryptic digests in rat tissue lysates. The expression levels of four different GPCRs were determined using chemically labeled synthetic standard peptides. Here, we demonstrate for the first time, that peptide immunoaffinity MS-based methods can render a reliable and quantitative analysis of multi-membrane spanning receptor molecules., (Copyright © 2013 Elsevier B.V. All rights reserved.)