Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400 ng/L, and the limit of detection was 14 ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.     

Rapid screening of lignans from Phyllanthus myrtifolius and stilbenoids from Syagrus romanzoffiana by HPLC-SPE-NMR

Introduction – Application of on‐line solid‐phase extraction (SPE) as an interface between HPLC and NMR has gained great improvement in solving sensitivity problems and signal interferences by the eluents. Objective – Rapid analysis and characterisation by HPLC‐SPE‐NMR and LC/MS of the arylnaphthalene‐type lignans present in Phyllanthus myrtifolius and the minor stilbenoids present in the polyphenol‐rich fraction from the ethanol extract of the seeds of Syagrus romanzoffiana. Methodology – Pretreatment of fractions by liquid–liquid partitioning, followed by Sephadex LH‐20 fractionation, was found very useful to facilitate the focusing and analysis of the polyphenolic fraction. HPLC‐DAD‐SPE‐NMR (400 MHz and 600 MHz) analysis was carried out using an Agilent 1100 liquid chromatography, followed by a Prospekt 2 automated solid‐phase extraction unit, containing 96 HySphere‐Resin GP cartridges (10 × 2 mm, 10–12 µm), which was connected to a 120 or 60 µL LC probe. Results – Seven arylnaphthalene‐type lignans from the chloroform‐soluble fraction of P. myrtifolius and nine stilbenoids from a polyphenol‐rich butanol‐soluble fraction of the seeds of S. romanzoffiana were characterised. Conclusion – HPLC‐SPE‐NMR associated with HR‐ESI/MS, which consumed only analytical amounts of partially purified mixtures, was demonstrated to be a good tool for rapid screening of both known and new natural products. Copyright © 2011 John Wiley & Sons, Ltd.     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

Predictable fold switching by the SARS‐CoV‐2 protein ORF9b

Extant fold‐switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer''s, and autoimmune disorders. Thus, predictive methods are needed to identify more fold‐switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high‐throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS‐CoV‐1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high‐throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.     

Linear measurement error models with restricted sampling

The relationship between nutrient consumption and chronic disease risk is the focus of a large number of epidemiological studies where food frequency questionnaires (FFQ) and food records are commonly used to assess dietary intake. However, these self-assessment tools are known to involve substantial random error for most nutrients, and probably important systematic error as well. Study subject selection in dietary intervention studies is sometimes conducted in two stages. At the first stage, FFQ-measured dietary intakes are observed and at the second stage another instrument, such as a 4-day food record, is administered only to participants who have fulfilled a prespecified criterion that is based on the baseline FFQ-measured dietary intake (e.g., only those reporting percent energy intake from fat above a prespecified quantity). Performing analysis without adjusting for this truncated sample design and for the measurement error in the nutrient consumption assessments will usually provide biased estimates for the population parameters. In this work we provide a general statistical analysis technique for such data with the classical additive measurement error that corrects for the two sources of bias. The proposed technique is based on multiple imputation for longitudinal data. Results of a simulation study along with a sensitivity analysis are presented, showing the performance of the proposed method under a simple linear regression model.     

Ontogeny and Metamorphic Patterns of Female Reproductive Organs of Ephedra sinica Stapf (Ephedraceae)

Ontogeny and metamorphic development of female reproductive organs in Ephedra sinica Stapf were surveyed. At the end of April, female cones began to initialize from the vegetative buds. Pollination occurred in mid-May and seeds matured at the beginning of July. The ontogenetic pattern of female reproductive organs of E.sinica is basically similar to that of E.　distachya L. The foliar nature of the outer envelope of the ovule in Ephedra is corroborated. Reduction of ovule number as a tendency of speciation in the genus is substantiated by the occurrence of tri-ovulate cones coupled with comprehensive characters of the genus. The metamorphic patterns as well as the leaf nature of the outer envelope indicate that female cones of Ephedra are compound while the female reproductive units of the ovulate cone have been reduced from secondary reproductive shoots similar to those of cordaites by phylogenetic transformation, fusion and reduction. Each fertile bract together with its axillary female reproductive unit composed the Bract Scale and Seed Scale Complex of Ephedra .     

Trends in solventless sample preparation techniques for environmental analysis

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.     

The real cost of sequencing: higher than you think!

Advances in sequencing technology have led to a sharp decrease in the cost of ''data generation''. But is this sufficient to ensure cost-effective and efficient ''knowledge generation''?