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Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A quantitative methodology using high resolution mass spectrometry was developed for the identification of organic compounds derived from wildfires in surface water samples. The methodology involves the use of solid-phase extraction (SPE) followed by detection using liquid chromatography-quadrupole time of flight-mass spectrometry (LC/Q-TOF-MS) for a group of fourteen chemical compounds (pyridine, benzene, naphthalene and biphenyl polycarboxylic acids). All compounds were successfully separated chromatographically using a reversed phase column and they were identified by accurate mass using the deprotonated species and their main fragment ions. The method produced excellent accuracies (>95%) and precisions (3-10%) for all the compounds studied. This methodology was successfully applied to the identification of fourteen compounds in runoff surface waters impacted by wildfires in Colorado in 2020. Concentrations of individual compounds ranging from 0.1 to 59.5 μg/L were found in wildfire impacted waters, with totals of ∼200 μg/L, thus showing these compounds as chemical tracers of wildfire events at significantly high concentrations. In addition, non-target analysis using chromatography patterns and mass spectrometry identification by MS-MS revealed other polycarboxylic acid isomers were also present in runoff surface water samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The issue of water contamination by heavy metal ions as highly persistent pollutants with harmful influence primarily on biological systems, even in trace levels, has become a great environmental concern globally. Therefore, there is a need for the use of highly sensitive techniques or preconcentration methods for the removal of heavy metal ions at trace levels. Thus, this research investigates a novel approach by examining the possibility of using pomegranate (Punica granatum) peel layered material for the simultaneous preconcentration of seven heavy metal ions; Cd(II), Co(II), Cr(III), Cu(II), Mn(II), Ni(II) and Pb(II) from aqueous solution and three river water samples. The quantification of the heavy metals was performed by the means of FAAS technique. The characterization of biomaterial was performed by SEM/EDS, FTIR analysis and pHpzc determination before and after the remediation process. The reusability study as well as the influence of interfering ions (Ca, K, Mg, Na and Zn) were evaluated. The conditions of preconcentration by the column method included the optimization of solution pH (5), flow rate (1.5 mL/min), a dose of biosorbent (200 mg), type of the eluent (1 mol/L HNO 3 ), sample volume (100 mL) and sorbent fraction (<0.25 mm). The biosorbent capacity ranged from 4.45 to 57.70 μmol/g for the investigated heavy metals. The practical relevance of this study is further extended by novel data regarding adsorbent cost analysis (17.49 $/mol). The Punica granatum sorbent represents a highly effective and economical biosorbent for the preconcentration of heavy metal ions for possible application in industrial sectors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Long term exposure to arsenic through consumption of contaminated groundwater has been a global issue since the last five decades; while from an alternate standpoint, arsenic compounds have emerged as unparallel chemotherapeutic drugs. This review highlights the contribution from arsenic speciation studies that have played a pivotal role in the progression of our understanding of the biological behaviour of arsenic in humans. We also discuss the limitations of the speciation studies and their association with the interpretation of arsenic metabolism. Chromatographic separation followed by spectroscopic detection as well as the utilization of biotinylated pull-down assays, protein microarray and radiolabelled arsenic have been instrumental in identifying hundreds of metabolic arsenic conjugates, while, computational modelling has predicted thousands of them. However, these species exhibit a variegated pattern, which supports more than one hypothesis for the metabolic pathway of arsenic. Thus, the arsenic species are yet to be integrated into a coherent mechanistic pathway depicting its chemicobiological fate. Novel biorelevant arsenic species have been identified due to significant evolution in experimental methodologies. However, these methods are specific for the identification of only a group of arsenicals sharing similar physiochemical properties; and may not be applicable to other constituents of the vast spectrum of arsenic species. Consequently, the identity of arsenic binding partners in vivo and the sequence of events in arsenic metabolism are still elusive. This resonates the need for additional focus on the extraction and characterization of both low and high molecular weight arsenicals in a combinative manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60 o C for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71-5.42, and an excellent linear relationship was obtained in the range of 0.25-500 pmol (R 2 ≥0.9993). The detection limit was 0.05-0.25 pmol. The intra- and inter-day precisions were 0.51-5.76% and 0.78-5.18%, respectively, and the average recovery was 90.03-99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tributylamine (TBuA) and triethylamine (TEtA) are the most commonly used ion pair reagents in ion pair chromatography especially for the analysis of oligonucleotides. In order to improve the understanding of the retention and separation mechanism of oligonucleotides in ion pair chromatography, it is important to understand the retention mechanism and the nature of interaction of these ion pair reagents with the stationary phase in the chromatographic column. Adsorption isotherm is helpful in evaluating such interactions, and subsequently predicting the retention mechanism. Alkylamines are very polar molecules which lack suitable chromophore and are commonly present in charged forms. Therefore, their determination and the subsequent acquisition of their adsorption isotherms using traditional liquid chromatography is very difficult. In this study, we first developed an analytical method for the determination of TBuA and TEtA in a typical chromatographic mobile phase (acetonitrile-water) and then used the same method to acquire the adsorption isotherms for tributylammonium acetate (TBuAA) and triethylammonium acetate (TEtAA). This method started with the conversion of the alkylammonium ions to free neutral forms by treating the sample with a strong base, followed by pentane-mediated extraction and finally the analysis of the extracts using gas chromatography-flame ionization detector (GC-FID). This three-step method was validated for parameters like range, linearity, intra-day and inter-day precision and accuracy, limit of detection and limit of quantitation. For the adsorption isotherms, the C18 column was first equilibrated with the solutions having different concentrations of alkylammonium ions and then stripped with eluent devoid of alkylammonium ions. Several stripping eluents were investigated and it was discovered that the eluent requirement could be decreased by the addition of sodium chloride. The effluents from the stripping phase were collected and analyzed using the developed analytical method to acquire the adsorption data. Under the investigated conditions, adsorption of TBuAA and TEtAA showed type III and type I isotherm behavior respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Efficient extraction is a vital step in mercury speciation. In this context, using 4-vinylbenzeneboronic acid and 9-vinylanthracene as functional monomers, a new magnetic adsorbent was fabricated according to one-pot hydrothermal approach. Various characterization results prove the as-prepared adsorbent presented abundant functional groups and saturation magnetism. Combining with magnetic solid phase extraction (MSPE), the adsorbent exhibited satisfactory entrapment performance towards different mercury species which had been pre-coordinated with dithizone to form metal-organic coordination. A series of parameters influencing the extraction performance were inspected in detail. Under the most beneficial conditions, sensitive and reliable approach to quantify trace methylmercury, ethylmercury, phenylmercury and inorganic mercury in aqueous samples was developed by the combination of HPLC/DAD. Limits of detection and precision located in the ranges of 0.012-0.074 µg/L and 2.5-9.8%, respectively. Recoveries with low, medium and high fortified contents in actual waters varied from 79.8 to 119%. Confirmatory experiments were performed to evidence the accuracy and reliability of established approach. In addition, a possible mechanism was suggested based on the chemical nature of analytes, extraction conditions and characterization results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Simple, effective and environment-friendly ways for remediating toxic metal pollution are necessary. In this study, the effect of different concentrations phosphate buffer solution (PBS) on removal efficiency of Pb and Zn in soil by solid phase microbial fuel cell (SMFC) was investigated. During 100 days of operation, the SMFC with 150 mM PBS generated the highest power density of 21.7 mW m -2 and the lowest internal resistance of 161 Ω. The addition of PBS can also increase soil conductivity and maintain a suitable pH for microbial activity. Furthermore, the removal rate of Pb and Zn in the SMFC with 150 mM PBS can reach 14.7% and 22.3%, respectively. The microbial community analyses demonstrated that Anditalea as an exoelectrogen in alkaline-saline conditions was significantly enriched in the SMFC with 150 mM PBS. This study provides an effective strategy for strengthening SMFC to remove toxic metals in soil., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The objective of this study was to investigate the effect of oximation reagents in simultaneous analysis of mono and di-saccharides using gas chromatography. Sugar oximation with O-ethylhydroxylamine separated all the mono- and di-saccharides while hydroxylamine and O-benzylhydroxylamine could make most of the saccharides separable except for xylose and arabinose. Resolution of xylose: arabinose, galactose: glucose, and fructose: galactose oximated by O-ethylhydroxylamine in DB-1ms column were 1.66, 2.15, and 6.19, respectively, which were above 1.5 and were officially acceptable for quantitative analysis according to the AOAC guideline. The applied method was then verified by the method validation parameters; LOD (0.011-0.02 mg/100 g), LOQ (0.032-0.061 mg/100 g), linearity (R 2  = 0.9991-1.0000) and precision (repeatability RSD: 1.4-3.3%, reproducibility RSD: 1.7-7.6%). The greatest amounts of xylose (19.03 ± 0.38 mg/100 g), maltose (6,274.48 ± 173.59 mg/100 g) were found in the oyster sauce, and the contents of glucose (10,565.00 ± 125.31 mg/100 g), galactose (170.40 ± 4.62 mg/100 g) were greatest in soybean paste., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Rationale: Saliva has been widely accepted as a more convenient alternative to serum or plasma in the field of clinical diagnosis. However, the detection of trace components in saliva has been a bottleneck problem. The aim of this work was to develop a highly sensitive and reliable method for simultaneously determining the trace steroid hormones including some with poor ionization efficiency in human saliva by liquid chromatography/tandem mass spectrometry (LC/MS)., Methods: Saliva was deproteinated by acetonitrile containing mixed isotope internal standards and extracted with methyl tert-butyl ether. The extraction solution was dried under a stream of nitrogen and the residue was derivatized using 50 mM O-ethylhydroxylamine hydrochloride in 80% methanol/water solution (v/v). The processed sample was determined by LC/MS in multiple reaction monitoring (MRM) mode., Results: The method was successfully established for the simultaneous quantification of seven steroid hormones in human saliva and showed excellent specificity and sensitivity. The limits of quantification (LOQs) of all steroid hormones were below 5 pg/mL, in particular, the LOQ of progesterone was as low as 0.15 pg/mL. The linear correlation coefficients (r) were greater than 0.9990 in the range of 2-200 pg/mL for T, DHEA, A4, P4, P5, and 17OHP4 and in the range of 5-500 pg/mL for 17OHP5. The intra-day and inter-day variability ranged from 1.86% to 7.83% and 1.95% to 10.4%, respectively. The recovery of the method ranged from 86.9% to 111.1% for all steroid hormones using three spiked concentrations., Conclusions: A novel LC/MS/MS method was developed for the simultaneous quantification of seven kinds of trace steroid hormones in human saliva. The results of the methodological study showed that the method exhibited excellent sensitivity and reliability for the evaluation of free steroid hormones in the human body. It is believed that this method could provide useful information of steroid hormone metabolism for auxiliary diagnosis of some endocrine disorders., (© 2021 John Wiley & Sons Ltd.)

The use of flame retardants, namely bis(2,3-dibromopropyl) phosphate (BDBPP) and tris(2,3-dibromopropyl) phosphate (TDBPP), in textile products such as curtains, carpets and sleeping clothes is banned in Japan under the 'Act on the Control of Household Products Containing Harmful Substances'. Herein, we developed a GC-MS based method to quantify these compounds with greater accuracy and safety than the current official method. For accurate and sensitive quantification, deuterated compounds, BDBPP-d 10 and TDBPP-d 15 , were used as surrogate standards. In consideration of the safety of the analyst, certain solvents and reagents used for the pretreatment that are carcinogenic or have a risk of explosion were replaced. For the extraction step, benzene was replaced by ethyl acetate, and for the methyl derivatization step, the reagent was changed from a self-prepared solution of diazomethane in ether to a solution of trimethylsilyl diazomethane in hexane, a safe and easy-to-use commercially available reagent. The calibration curves were liner in the range of 0.5-8.0 μg/mL for both methylated BDBPP (BDBPP-Me) and TDBPP. The detection limit was 0.05 μg/g for BDBPP-Me and 0.3 μg/g for TDBPP, which is sufficiently low compared to the current detection limits of 10 μg/g for BDBPP-Me and 8 μg/g for TDBPP. The recoveries in various curtain material were 66-108% and relative standard deviations were 1.2-10.2% when 5 μg BDBPP and TDBPP were added to 0.5 g of samples. Thus, the developed method is applicable to textile products of various materials.

Background: The biologic scaffolds derived from decellularized tissues and organs have been successfully developed in a variety of preclinical and/or clinical studies., Objective: The new decellularized liver-regenerative 3D printing biomaterials were designed and prepared for cell-based liver therapies., Methods: An extraction process was employed to remove the tissue and cellular molecules from porcine liver via pretreatment of supercritical fluid of carbon dioxide (ScCO2). Varying porosities of the decellularized liver tissues were created using papain-containing reagent treatments after ScCO2., Results: The resulting liver-regenerative 3D printing biomaterials of decellularized liver collagen scaffolds were characterized by Fourier transform infrared spectroscopy, thermo-gravimetric analysis, differential scanning calorimetry and scanning electron microscopy., Conclusions: The decellularized liver collagen scaffolds with good thermal stability (>150 °C) were obtained and employed as liver-regenerative 3D printing biomaterials for cell-based liver therapies.

In this paper, a novel and stable fluorescent reagent, quinoline-2,3-dicarbaldehyde (QDA), is synthesized as a probe to detect ammonium in natural water. Ammonium reacts with QDA in the presence of SO 3 2- and Ca 2+ to form a fluorescence product, which has maximum excitation and emission wavelengths at 429 nm and 518 nm. The concentration of reagents, the reaction temperature, the reaction time, and the pH in the final solution are investigated and optimized. The interferences of typical organic nitrogen and inorganic compounds are evaluated, and results prove that most volatile amines have little or negligible effect. Under the optimized conditions, this method provides a limit of detection of 0.065 μmol L -1 , a calibration range of 0.216-9 μmol L -1 , and reproducibility (with a relative standard deviation) of 1.9% for 1.5 μmol L -1 ammonium. For water sample analysis, the proposed method provides comparable results to those of the conventional o -phthalaldehyde method but has longer reagent stability (42 days).

Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete 'infectome' (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple 'blank' negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae , Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

Herein, uric acid@Ti 3 C 2 quantum dots (UA@Ti 3 C 2 QDs) were synthesized via a microwave-assisted strategy on the basis of acid etching and stripping. The UA@Ti 3 C 2 QDs have bright blue emission. Intriguingly, the fluorescence emission of the UA@Ti 3 C 2 QDs was significantly quenched after the addition of 2,4,6-trinitrophenol (TNP), due to inner-filter effect (IFE). Based on these findings, a novel environmentally friendly and water-soluble fluorescence probe based on UA@Ti 3 C 2 QDs was demonstrated for the sensitive and selective detection of TNP. The method presented a wide linear range for TNP detection in the 0.01-40 μM range, with a low detection limit of 9.58 nM. Furthermore, the probe was successfully used for the sensitive detection of TNP in real water and smartphone-based colorimetric (SPBC) detection of TNP on surfaces with the linear range from 10.0 to 100.0 ng. On the whole, this work provides an effective strategy for the synthesis of UA@Ti 3 C 2 QDs and an alternative fluorescence probe for detecting TNP both on surface and in solution.

Sorption of PFASs onto surfaces of laboratory materials has been frequently reported. Due to the often complex and poorly understood nature of such sorption, workarounds have often included use of whole samples only, accompanied by sample vessel rinsing to desorb active surfaces. The resulting methods tend to require considerable sample preparation times and preclude typical activities such as aliquoting and dilution of water samples prior to extraction. This manuscript reports an approach for PFAS analysis which uses subsampling of water matrices from vessels including centrifuge tubes and autosampler vials, through the optimized use of solvent to reduce PFAS retention on subsampling vessels. Online solid phase extraction (SPE) using a weak anion exchange resin is then used to concentrate sample aliquots to improve sensitivity and allow for removal of matrix interferences. With the technique of ultra performance liquid chromatography (UPLC) coupled to isotope dilution tandem mass spectrometry, statistically based quantitation limits ranged from sub ng/L to single digit ng/L for carboxylate, sulfonate, and sulfonamide PFASs analytes from C 4 to C 12 . Linear calibration ranges were from 0.25 to 4000 ng/L. Matrix effects relevant for drinking water treatment studies, such as cations, organic carbon, and competing PFAS compounds, were evaluated and found to not impact method performance within QC criteria consistent with study data quality objectives., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Background: There is a need for a reference material to support the development and ensure the quality of immunoassays for human AMH. A batch of ampoules, coded 16/190, containing lyophilised recombinant AMH was evaluated in a WHO Collaborative Study. The aims of the study were to determine the AMH content in terms of the calibration of each immunoassay method, to predict long-term stability and to assess the suitability of the preparation to calibrate AMH immunoassays., Methods: Study participants were asked to report the AMH content of specific dilutions of coded ampoules of 16/190 and a comparator preparation containing approximately half the AMH content. In each assay, participants also reported the AMH content of 22 patient samples to assess commutability. A robust all-laboratory geometric mean of the content estimates was determined using the laboratory geometric mean estimates. Commutability was assessed using a difference in bias approach. Stability was predicted by the measurement of thermally accelerated degradation samples., Results: Seven laboratories performed twenty-one immunoassay method-platform combinations, sixteen of which provided data which met the validity criteria, giving a consensus geometric mean estimate of AMH content of 511 ng/ampoule (95% CI, 426-612, n = 16, GCV 42%) and a robust geometric mean of 489 ng/ampoule. By contrast, the GCV% for the all-laboratory geometric mean of the relative content estimates for the comparator sample to 16/190 was 12%. Commutability was assessed using 20 of the 22 representative patient samples. Of the valid assays, 16/190 was within the limits of acceptable commutability for 6 methods, partially commutable for a further 3 methods and non-commutable when measured by 7 methods. The preparation was predicted to be highly stable when stored at - 20 °C., Conclusion: The majority of methods met the validity criteria. Content estimates showed a high between-method variability, yet assays exhibited a similar proportionality of response as demonstrated using the comparator sample. 16/190 was commutable in some but not all methods. On the basis of these results, it was agreed by the WHO Expert Committee on Biological Standardization to establish 16/190 as a WHO Reference Reagent for AMH with a content defined by consensus immunoassay of 489 ng/ampoule.

The ability of an enzyme linked immunosorbent assay (ELISA) to measure the concentration of charge variants of a monoclonal antibody (mAb) biotherapeutic has been investigated. Percentage recovery was found to be in the range of 80%-120% with inter and intra assay variation between 5% and 15%. Linear regression analysis of ELISA output at different dilutions yielded a good fit (R 2  > 0.98). Assay output was not hindered by the presence of serum proteins and other analytes such as GCSF, demonstrating acceptable precision and specificity. It was concluded that the charge variants of the mAb can be accurately quantified by ELISA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: using the urinary cotinine biomarker to verify the occurrence of green tobacco sickness in workers who cultivate Burley tobacco., Method: paired case-control study, based on smoking status and on the 1:4 ratio, with participation of 20 case workers and 91 controls. Data collection included household surveys and urine collection for cotinine examination. Student's T-Test, the Mann-Whitney test, Pearson's chi-square or Fisher's exact tests were used., Results: of the 23 suspected cases, 20 showed elevated levels of cotinine, signs and symptoms of headache, skin irritation, nausea, sickness and general malaise, especially in the morning. Most had worked with tobacco that was wet from the morning dew and when the weather was warm., Conclusion: there are signs suggestive of green tobacco sickness in Burley tobacco workers. The action of health professionals is necessary for the development of health promotion and preventive actions addressing work-related illness.

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device 1 . A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters 2-5 . This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents 6 . One solution is to limit the rate of release of reagents into the liquid 7-10 . However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.

Multivariate data analysis feasibility for the evaluation of Brazilian stingless bee honey (SBH) by pollen spectrum, bioactive compounds content, physicochemical, antioxidant and antimicrobial analysis was investigated. Levels of total and individual phenolics content were analyzed by HPLC-PDA. The antioxidant capacity was performed by 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH), oxygen radical absorption capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays. The total phenolic compounds from the thirty-two SBH was positively correlated with the antioxidant capacity. Bioactive compounds such as p-coumaric acid, quercetin, and hesperetin were identified in all the samples. Brazilian SBH shows more effective antibacterial activity against Gram-negative bacteria (E. coli and S. Typhimurium) compared to Gram-positive ones. Results also revealed that SBH could reach up to 45% higher antioxidant and biological activities than the traditional Apis mellifera honey. Chemometrics shows that chemical and biological properties of SBH have a strong relationship with the pollen botanical origin. Principal component analysis (PCA) grouped the honey into three categories with predominant pollen from Verbenaceae, Asteraceae and Sapindaceae families, confirming that SBH belonging to the same floral origin present similar characteristics., (Copyright © 2019. Published by Elsevier Ltd.)

Soft chemical ionization mass spectrometry techniques, particularly the well-established proton transfer reaction mass spectrometry, PTR-MS, and selected ion flow tube mass spectrometry, SIFT-MS, are widely used for real-time quantification of volatile organic compounds in ambient air and exhaled breath with applications ranging from environmental science to medicine. The most common reagent ions H 3 O + , NO + , or O 2 +• can be selected either by quadrupole mass filtering from a discharge ion source, which is relatively inefficient, or by switching the gas/vapor in the ion source, which is relatively slow. The chosen reagent ions are introduced into a flow tube or flow-drift tube reactor where they react with analyte molecules in sample gas. This article describes a new electrostatic reagent ion switching, ERIS, technique by which H 3 O + , NO + , and O 2 +• reagent ions, produced simultaneously in three separate gas discharges, can be purified in post-discharge source drift tubes, switched rapidly, and selected for transport into a flow-drift tube reactor. The construction of the device and the ion-molecule chemistry exploited to purify the individual reagent ions are described. The speed and sensitivity of ERIS coupled to a selected ion flow-drift tube mass spectrometry, SIFDT-MS, is demonstrated by the simultaneous quantification of methanol with H 3 O + , acetone with NO + , and dimethyl sulfide with O 2 +• reagent ions in single breath exhalations. The present ERIS approach is shown to be preferable to the previously used quadrupole filtering, as it increases analytical sensitivity of the SIFDT-MS instrument while reducing its size and the required number of vacuum pumps.

Nucleotide sequence reagents are verifiable experimental reagents in biomedical publications, because their sequence identities can be independently verified and compared with associated text descriptors. We have previously reported that incorrectly identified nucleotide sequence reagents are characteristic of highly similar human gene knockdown studies, some of which have been retracted from the literature on account of possible research fraud. Because of the throughput limitations of manual verification of nucleotide sequences, we developed a semi-automated fact checking tool, Seek & Blastn, to verify the targeting or non-targeting status of published nucleotide sequence reagents. From previously described and unknown corpora of 48 and 155 publications, respectively, Seek & Blastn correctly extracted 304/342 (88.9%) and 1066/1522 (70.0%) nucleotide sequences and a predicted targeting/ non-targeting status. Seek & Blastn correctly predicted the targeting/ non-targeting status of 293/304 (96.4%) and 988/1066 (92.7%) of the correctly extracted nucleotide sequences. A total of 38/39 (97.4%) or 31/79 (39.2%) Seek & Blastn predictions of incorrect nucleotide sequence reagent use were correct in the two literature corpora. Combined Seek & Blastn and manual analyses identified a list of 91 misidentified nucleotide sequence reagents, which could be built upon through future studies. In summary, incorrect nucleotide sequence reagents represent an under-recognized source of error within the biomedical literature, and fact checking tools such as Seek & Blastn may help to identify papers and manuscripts affected by these errors., Competing Interests: The authors have declared that no competing interests exist.

Background noise in metagenomic studies is often of high importance and its removal requires extensive post-analytic, bioinformatics filtering. This is relevant as significant signals may be lost due to a low signal-to-noise ratio. The presence of plasmid residues, that are frequently present in reagents as contaminants, has not been investigated so far, but may pose a substantial bias. Here we show that plasmid sequences from different sources are omnipresent in molecular biology reagents. Using a metagenomic approach, we identified the presence of the (pol) of equine infectious anemia virus in human samples and traced it back to the expression plasmid used for generation of a commercial reverse transcriptase. We found fragments of multiple other expression plasmids in human samples as well as commercial polymerase preparations. Plasmid contamination sources included production chain of molecular biology reagents as well as contamination of reagents from environment or human handling of samples and reagents. Retrospective analyses of published metagenomic studies revealed an inaccurate signal-to-noise differentiation. Hence, the plasmid sequences that seem to be omnipresent in molecular biology reagents may misguide conclusions derived from genomic/metagenomics datasets and thus also clinical interpretations. Critical appraisal of metagenomic data sets for the possibility of plasmid background noise is required to identify reliable and significant signals.

Reagent-derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the 'kitome' (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents. In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing. Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads. We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low-biomass microbiome studies and producing more robust and reliable data. SIGNIFICANCE AND IMPACT OF THE STUDY: Reagent contamination with microbial DNA is a major problem in microbiome studies of low microbial biomass samples. Levels of such contaminating DNA often outweigh what is present in the sample and heavily confound subsequent data analysis. Previous studies have suggested this contamination is primarily derived from DNA extraction kits. Here, we identified the PCR master mix as the primary source of contamination, and showed that enzymatic removal of the contamination drastically reduced the blank signal and improved precision. Decontamination of PCR master mixes may have the potential to improve the sensitivity and accuracy of low-biomass microbiome studies., (© 2018 The Society for Applied Microbiology.)

Introduction: Multi-site tobacco cessation trials could benefit from remote biochemical verification for tobacco use without invasive, time-consuming, or expensive collection processes. To the authors' knowledge, there have been no previous studies examining the predictive validity of oral fluid swabs for the detection of cotinine levels with samples collected off-site and mailed for on-site interpretation., Methods: Tobacco users were recruited through an online survey and participants who met the initial eligibility criteria were invited to take part. Those who elected to enroll provided two positive iScreen Oral Fluid Device (OFD) cotinine test samples during an in-office visit. One sample was used as a control and stored in a temperature-regulated location, while the other was mailed from one of ten surrounding counties. Mailing method and time from collection to mailing were varied, and results were assessed against control samples., Results: Twenty tobacco users enrolled in the study. Participants ranged in age from 18 to 31 (M = 16.45, SD = 1.54). Several types of tobacco use were reported, with electronic cigarettes the most commonly reported product. None of the mailed sample interpretations changed from pre- to post-mailing, with up to twenty-one days from sample collection to results confirmation., Conclusions: Results indicate that the use of mailed oral swabs may be an easy to use, reliable, and low-cost option for the detection of cotinine in tobacco users when in-person collection is not feasible. Test result interpretations were found to be unchanged after mailing, and after extended post-collection time gaps., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The search for biosignatures on spaceflight missions requires in situ instrumentation capable of highly selective and sensitive organic analyses. To this end, CE-LIF is a uniquely promising technique, capable of determining the type, abundance, and chirality of amino acids present in environmental samples at nanomolar concentrations. However, this type of assay requires several reagents that have not yet been used on spaceflight missions. A key concern, particularly for future missions to Europa, is the survivability of these critical components for CE separation and LIF detection under high levels of radiation. Here we present an investigation of the chemical stability of the reagents and associated fused silica capillary after a total ionizing dose of 300 krad, exceeding the predicted total ionizing dose for the potential Europa Lander Mission payload by two-fold. Neither the fused silica capillary nor the fluorescent dye (5-carboxyfluorescein succinimidyl ester) showed significant change in performance following irradiation. Following the irradiation of the pre-mixed background electrolyte, both migration time and resolution were affected. However, when the reagents (sodium tetraborate, sodium taurocholate, and γ-cyclodextrin) and the acetonitrile solution were irradiated separately and mixed afterwards, there was no change in the separation performance., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The study of cellular Ca 2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca 2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca 2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Real-time low to sub parts-per-trillion (ppt v ) vapor detection of some organophosphorous compounds (OPCs) is demonstrated with an atmospheric flow tube-mass spectrometer. The chemical species investigated included dimethyl methylphosphonate, triethyl phosphate, and tributylphosphate. The atmospheric flow tube provides ambient chemical ionization with up to several seconds of ionization time. With sensitivities in the parts-per-quadrillion (ppq v ) range, there are many background contaminants competing for charge with the target analytes. Initially, the OPCs were not observable in direct room air analysis, presumably due to other trace components possessing higher proton affinities. However, the addition of a trialkylamine as a dopant chemical served to provide a single reagent ion that also formed a proton-bound heterodimer with the OPCs. These asymmetric proton-bound dimers had sufficiently high hydrogen bond energy to allow the cluster to remain intact during the analysis time of several seconds. Changes in stability were observed for some of these asymmetric proton-bound dimers with a shorter half-life for adducts with a larger proton affinity differences between the amine and the OPC. Detection levels approaching low ppt v to high ppq v were correlated by three different methods, including use of a permeation tube, direct injection of a fixed mass into the sample air flow, and calculations based upon signal intensity ratios, reaction time, and an estimated reaction rate constant. A practical demonstration showed real-time monitoring of a laboratory environment initially with low ppt v levels of vapor observed to decay exponentially over about an hour while returning to baseline levels.

Purpose: The protective function of the intestinal mucosa largely depends on carbohydrate moieties that as a part of glycoproteins and glycolipids form the epithelial glycocalyx or are secreted as mucins. Modifications of their expression can be induced by an altered intestinal microenvironment and have been associated with inflammatory disorders and colorectal cancer. Given the influence of dietary factors on the gut ecosystem, here we have investigated whether a long term feeding on a starch-rich diet can modulate the glucidic profile in the colonic mucosa of rats., Methods: Animals were divided into two groups and maintained for 9 months at different diets: one group was fed a standard diet, the second was fed a starch-enriched diet. Samples of colonic mucosa, divided in proximal and distal portions, were processed for microscopic analysis. Conventional stainings and lectin histochemistry were applied to identify acidic glycoconjugates and specific sugar residues in oligosaccharide chains, respectively. Some lectins were applied on adjacent sections after sialidase/fucosidase digestion, deacetylation, and oxidation to characterize either terminal dimers or sialic acid acetylation., Results: An increase in sulfomucins was found to be associated with the starch-enriched diet that affected also the expression of several sugar residues as well as fucosylated and sialylated sequences in both proximal and distal colon., Conclusions: Although the mechanisms leading to such a modulation are at present unknown, either an altered intestinal microbiota or a dysregulation of glycosylation patterns might be responsible for the types and distribution of changes in the glucidic profile here observed.

Background and Objectives: Unlike cigarette smoking cessation, waterpipe tobacco smoking cessation is relatively understudied. The objective of this randomized clinical trial was to examine the efficacy of contingency management (CM) for promoting initial waterpipe smoking abstinence., Methods: The study used a two-group, repeated measures design. Participants attended 10 visits (two visits per week, on Mondays and Thursdays) across 5 weeks. Thirty-nine adult waterpipe tobacco users who did not smoke cigarettes and were not planning on quitting waterpipe tobacco smoking were randomly assigned to either the contingent (n = 19) or non-contingent (n = 20) groups. Contingent group received monetary rewards based on negative salivary cotinine results. Earning rewards started at $14 and increased by $.50 with each subsequent negative sample for a maximum $192.50. Non-contingent group earned rewards independent of salivary cotinine results. Prolonged abstinence was defined as having negative salivary cotinine results for eight or more visits (two lapses were allowed); and 7-day point prevalence was defined as having negative salivary cotinine results at visit 9 and 10 (final week)., Results: The prolonged abstinence rate in the contingent and non-contingent groups were 42.1% and 5.0%, respectively, (p = .008). The 7-day point prevalence in the contingent and non-contingent were 47.4% and 5.0%, respectively, (p = .003)., Discussion and Conclusions: Rewards contingent on biochemically verified abstinence promote initial waterpipe tobacco cessation. This is useful information for consideration in future cessation programs for waterpipe smokers., Scientific Significance: CM strategy may have potential benefit in addressing waterpipe tobacco smoking in non-treatment seeking adults. (Am J Addict 2018;27:202-209)., (© 2018 American Academy of Addiction Psychiatry.)

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

In nutrition studies, para-aminohippuric acid (PAH) is a marker frequently used to measure blood flow in pigs, which is essential for estimating portal-drained viscera (PDV) flux of nutrients. The aim of this study was to evaluate the PAH analytical method by means of qualimetric statistical procedures to estimate the matrix effect and the accuracy and limits of quantitation of the method. Net PDV flux of nutrients was determined in five multi-catheterized pigs using water, plasma or commercial serum as standard matrix. A proportional systematic error due to matrix effect was found for plasma and serum. Mean recovery was 99.4%, and intra- and inter-day precision of the method was 2.4% and 3.8% relative standard deviation, respectively. The limit of quantification was 0.22 mg PAH/l. Use of water for the PAH standard curves underestimated portal blood flow compared with PAH standards prepared with plasma or commercial serum (706, 954 and 927 ml/min; P<0.05, respectively). Consequently, PDV O2 consumption, glucose and amino acids fluxes were underestimated by 33% (P<0.001). In conclusion, our results stress the importance of using plasma from pigs not infused with PAH or alternatively commercial pig serum to prepare PAH standards to determine blood flow in pigs to avoid underestimation of blood flow.

Chemical 'spot' tests are a presumptive illicit drug identification technique commonly used by law enforcement, border security personnel, and forensic laboratories. The simplicity, low cost, and rapid results afforded by these tests make them particularly attractive for presumptive identification globally. In this paper, we review the development of these long-established methods and discuss color test recommendations and guidelines. A search of the scientific literature revealed the chemical reactions occurring in many color tests are either not actively investigated or reported as unknown. Today, color tests face many challenges, from the appearance of new psychoactive substances to concerns regarding selectivity, sensitivity, and safety. Advances in technology have seen color test reagents used in digital image color analysis, solid sensors, and microfluidic devices for illicit drug detection. This summarizes current research and suggests the future of presumptive color testing., (Copyright © 2017 John Wiley & Sons, Ltd.)

A discussion of the definition, advantages, and issues with the formation of ions in the transition region between an electrospray ionization (ESI) source and the ion optics of a mass analyzer is presented. The various types of ions formed in the so-called in-source collision-induced dissociation (IS-CID) process are illustrated. Applications of IS-CID with single-stage mass analyzers, such as structure elucidation and quantitation, are demonstrated. The discussion is illustrated by examples of the in-source fragmentation of ginkgolides, which are marker compounds found only in Ginkgo biloba. Supercritical fluid chromatography (SFC) with non-aqueous eluents was used to achieve a fast resolution of the ginkgolides without the hydrolysis reactions possible with aqueous high-performance liquid chromatography (HPLC) eluents. In-source ion generation occurs at relatively high pressures (ca. 1-3 torr) compared to the low pressure normally observed in collision chambers of tandem mass spectrometry (MS/MS). As a result, the fragmentation process is complex and often generates ions other than the fragments observed with classic CID or the same ions at different intensities. The objective of the current tutorial is to illustrate the conditions under which single-stage, quadrupole or time-of-flight mass analyzers with electrospray or in-air (direct analysis in real time; DART) ionization can be used for quantitation and structure elucidation in a manner similar to that observed with MS/MS. While the low m/z (≤ [M±H] ± ) ions formed in-source often duplicate the ions observed in MS/MS systems, it is the focus of this discussion to illustrate the utility of in-source generated fragment ions that may not be observed or observed at different intensities than in the collision cells of MS/MS instruments., (Copyright © 2017 John Wiley & Sons, Ltd.)

Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding.

Soil-transmitted helminths (STHs) are a major health concern globally. Infection is mostly through contact with contaminated water, food or soil. Therefore to break the cycle of viable transmission STH eggs must be quantitatively detected in the environment. The effect of different reagents on the viability of Ascaris suum eggs during laboratory detection and quantification was assessed and different incubation solutions compared. Sulphuric acid gave a slightly higher recovery percentage of viable eggs (91.2%) than distilled water (90.0%) and 0.5% formalin (87.6%), although the difference was not statistically significant (p > 0.05). Acetoacetic acid, ethyl acetate, ammonium bicarbonate, zinc sulphate, magnesium sulphate and Tween 80, are reagents widely used in test protocols for the detection and quantification of STH eggs. Eggs were exposed to these reagents for different time durations. Acetoacetic acid resulted in the highest loss of viability (3.4 ± 0.7% viable), while magnesium sulphate resulted in the least effect (88.5 ± 1.2% viable). In conclusion the use of the selected reagents in the detection of these eggs was found to affect the viability of exposed eggs, especially during prolonged exposures. Therefore we recommended that eggs be exposed for ≤5 minutes, to reduce the risk of viability loss.

Streptococcus pneumoniae is a major human pathogen, causing around 1.6 million deaths worldwide each year. By optimizing a resazurin-based assay to detect S. pneumoniae growth in 384-well microplates, we developed a new high-throughput screening (HTS) system for the discovery of antipneumococcal molecules, which was unsuccessful using conventional absorbance measurements. Before applying our protocol to a large-scale screen, we validated the system through a pilot screen targeting about 7,800 bioactive molecules using three different S. pneumoniae serotypes. Primary screenings of a further 27,000 synthetic small molecules facilitated the identification of 3-acyl-2-phenylamino-1,4-dihydropquinolin-4-one (APDQ) derivatives that inhibited growth of S. pneumoniae with MIC 90 values <1 μM (0.03-0.81 μM). Five selected APDQ derivatives were also active against Staphylococcus aureus but neither Klebsiella pneumoniae nor Pseudomonas aeruginosa, suggesting that APDQ may act specifically against Gram-positive bacteria. Our results both validated and demonstrated the utility of the resazurin-based HTS system for the identification of new antipneumococcal molecules. Moreover, the identified new antipneumococcal molecules in this study may have potential to be further developed as new antibiotics.

Resazurin microtitre assay (RMA) has been successfully used to detect minimal inhibitory concentrations (MICs) of both first-line and several second-line drugs in drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). In this study, we firstly compared prothionamide (PTH) susceptibility testing of Mycobacterium tuberculosis (MTB) using resazurin microtitre assay (RMA) and MGIT. Overall, the sensitivity and specificity of RMA for detecting PTH susceptibility was 96.5% [95% confidence interval (CI): 91.7-100.0] and 93.2% (95% CI: 89.6-96.8) respectively. In addition, the median time to positivity was significantly shorter for RMA than for the automated MGIT 960 (RMA, 8 days [range: 8-8 days] vs MGIT, 10.1 days, [range: 5.0-13.0]; P < 0.01). Concordance rate for MICs between RMA and MGIT for PTH-resistant group was 64.3% (95% CI: 46.5-82.0), which was significantly lower than that of PTH-susceptible group (85.9%, 95% CI: 78.8-93.0; P= 0.01). In conclusion, our data demonstrated that RMA can be used as an acceptable alternative for determination of PTH susceptibility with shorter turn-around time. When compared with MGIT 960, RMA method was prone to produce higher MICs for PTH-resistant MTB strains.

This assay facilitates the immunometric determination of assay-associated reagents including the capture and detection antibodies as well as the analyte (i.e., antigen or antibody). It can precede the development of a sandwich enzyme-linked immunosorbent assay (ELISA) in which optimal antibody concentrations are applied for the quantitative measurement of the antigen. This protocol describes the materials and equipment required for the measurement of chromogenic substrate development; however, it can be adapted for use with chemiluminescent- and fluorescent-labeled reporters., (© 2017 Cold Spring Harbor Laboratory Press.)

The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial 'decorin' and 'biglycan' were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of 'decorin' and 'biglycan' on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers' funds and time., (© 2017 The Author(s).)

Background: The relationships between the hydrophilic-lipophilic balance (HLB) of antioxidants (AOs) and their distributions and efficiencies in emulsions are not fully understood. Recent reports indicate that, for series of homologous antioxidants of different hydrophobicity, the variation of their efficiency with the HLB of the AO increases with the alkyl chain length up to a maximum (C 3 -C 8 ester) followed by a decrease (cut-off effect)., Results: We determined the distributions of a series of caffeic acid derivatives in intact soybean emulsions by employing a specifically designed chemical probe located in the interfacial region of the emulsion. We also determined the AO efficiencies in the very same emulsions. We demonstrate that the variation of the percentage of AO in the interfacial region of soybean oil-in-water emulsions with the AO HLB parallels that of their antioxidant efficiency., Conclusion: The results provide physical evidence that the variations in the efficiency of homologous series of antioxidants in emulsions are the result of differences in their distribution. The results confirm that, with other things being equal, there is a direct relationship between the percentage of AO in the interfacial region of the emulsions and their efficiency, providing a natural explanation, based on molecular properties, of the cut-off effect. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copolymerized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside time and resource saving.

In this work we propose a rapid and efficient method for the joint determination of nucleosides and nucleotides in dairy and non-dairy baby foods based on hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of diethylammonium (DEA) as a hydrophilic ion-pairing reagent (IP-HILIC-MS/MS). Sample treatment of the baby food included dilution with water and centrifugal ultrafiltration (CUF) with an additional washing step that notably improved the global performance of the process. Later dilution of the extract with acetonitrile allowed adequate separation in the HILIC system. With the proposed treatment, we obtained extraction recoveries higher than 80% and, additionally, no matrix effects were observed. The CUF-IP-HILIC-MS/MS method was validated according to the 2002/657/EC decision and was used for the quantification of nucleotides and nucleosides in sixteen samples of commercial baby foods., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid., Methods: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation., Results: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid., Conclusions: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The most specific method of the recording of the rate offree radical reactions is the method of electron paramagnetic resonance (EPR) spectroscopy, but it is rarely used in applied biology due to expensive equipment and complexity of the execution of measurements. However chemists have found a number of colored organic radicals which lose the coloring under transition into diamagnetic form. In the given paper there are presented results of our studies on the development of methods for the assessment of oxidant equilibrium in biological media with a use of stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and cation-radicals of N,N-diethyl-p-phenylenediamine (DEPPD). We have developed the new modification of DPPH test, replacing methanol-based incubation medium by non-ionic detergent solution, compatible with native blood serum. Modified DPPH test conserved typical biphasic kinetics of the origin variant, had the similar sensitivity to model antioxidants (IC values 49, 38 and 13 mkMfor ascorbate, a-tocopherol and quercetine, correspondingly) and was applied in experiments on laboratory animals treated with nano- and ionic silver, carbon nanotubes, microfine coal and electrolytic dust. We have tried also the assay of serum lipid hydroperoxides based on Fe-initiated DEPPD oxidation (Alberti et al., 2000). The comparison of kinetics of DEPPD oxidation in model (HO/Fe) and biologic (rat serum/Fe) systems, before and after Fe addition, seems to be an evidence that ceruloplasmin (CP) was involved in the resulting process, but failed to determine its polynomial kinetics, at least for the rat serum and DEPPD excess. The use of CP monoclonal antibodies seems to be the best way for the clarification of the mechanism of this reaction.