•Amended methods improved detection across classes, most notably for nucleosides.•Detection limits improved up to 20-fold by screening benzoylation conditions.•New LC derivatization protocols ...compatible with at least four biological systems.•Aliphatic hydroxyl group benzoylation impacted by base concentration and choice.
LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e., specific bases), other hydroxyl groups may be derivatized too. Previous studies have examined BzCl concentration, reaction times, and reaction temperatures for derivatization of amines and phenols for LC-MS analysis; however, use of different bases, base concentration, and extending to conditions to hydroxyl groups for LC-MS analysis has not been well-studied. To address this understudied area and identify reaction conditions for both amino and hydroxyl groups, we performed a systematic study of reaction conditions on multiple classes of potential targets. For selected derivatization methods, detection limits and performance in a variety of biological matrices were assessed. Results highlight the importance of tailoring derivatization methods for a given application as they varied by molecule and/or molecule class. Compared to the standard BzCl method commonly used, alternative methods were identified to better derivatize challenging analytes (glucosamine, choline, cortisol, uridine, cytidine) with detection limits reaching 1100, 9, 38, 170, and 67 nM compared to undetectable, 170, 86, 1000, and 86 nM respectively. Sub-nanomolar detection limits were achieved for norepinephrine with alternative derivatization approaches. Improved derivatization methods for several classes and molecules including nucleosides, steroids, and molecules containing hydroxyl groups were also identified.
**Please note this is an unedited paperback reprint of the hardback, originally published in 2003**
The British system of universal development control celebrated its 50th anniversary in 1997. ...Remarkably, the system has survived more or less intact but the experience of the 1980s has left large questions unanswered about the relevance and effectiveness of the system. This book traces the history of the development control system in Britain from early modern times to the present day.
•Western blotting is performed from nanogram scale samples.•Protein samples are separated and captured on a membrane in 15 minutes.•Fluorescent antibodies are used to simultaneously detect ladder and ...analyte.
Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 – 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, β-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.
Having Your Say Shackleton, J. R; Oderberg, David S; Booth, Philip ...
2021, 2021-07-01
eBook
Today should be a Golden Age for free speech - with technology providing more ways of communicating ideas and opinions than ever before. Yet we're actually witnessing a growing wave of restrictions ...on freedom of thought and expression. In Having Your Say a variety of authors - academics, philosophers, comedians and more - stress the fundamental importance of free speech, one of the cornerstones of classical liberalism. And they provide informed and incisive insights on this worrying trend, which threatens to usher in a new, intolerant and censorious era.
This paper examines the development of Catholic social thought and teaching on the responsibilities of business. It begins with a natural law and Thomistic framework and shows how the general ...principles are applied to practical situations, firstly in the work of the late scholastics and, secondly, through the social encyclical tradition. Changes in emphasis are noted in the post-war period. The paper also explores how the challenges of corruption and lobbying have been viewed in the tradition. Despite mention of these topics by Pope Francis, we conclude that they are under-explored areas. As such, we set out a framework for consideration of these topics using the approach presented in the earlier part of the paper.
The importance of private property has been controversial in Catholic social thought and teaching down the ages. The approach of the Catholic Church to the question of property rights has been ...discussed by a number of Austrian-school authors, though their focus has been on teaching documents from 1891 to 1931. During that period, and up to the mid-1960s, the modern teaching of the Catholic Church on the right to property tended to be extremely positive. Since that time, ambiguities have crept in, and Pope Francis in particular has given the impression of being lukewarm towards the right to property. We discuss whether Pope Francis’s position is radical within the tradition of Catholic social teaching and conclude that it is not especially so, though his emphasis is more negative than that of previous popes and his reasoning not always sound. It is also found that the Catholic Church has left undeveloped the link between private property, economic development and environmental outcomes despite the interest of the Church in poverty and environmental degradation. This, together with the lack of discussion of the exceptions to the general rule of support for private property, is a real omission in Catholic Church teaching.
Joubert syndrome (JBTS) is an incurable multisystem ciliopathy syndrome. The most commonly mutated gene in JBTS patients with a cerebello-retinal-renal phenotype is CEP290 (alias JBTS5). The encoded ...CEP290 protein localises to the proximal end of the primary cilium, in the transition zone, where it controls ciliary protein composition and signalling. We examined primary cilium structure and composition in fibroblast cells derived from homozygous and compound heterozygous JBTS5 patients with nonsense mutations in CEP290 and show that elongation of cilia, impaired ciliogenesis and ciliary composition defects are typical features in JBTS5 cells. Targeted skipping of the mutated exon c.5668 G > T using antisense oligonucleotide (ASO) therapy leads to restoration of CEP290 protein expression and functions at the transition zone in homozygous and compound heterozygous JBTS5 cells, allowing a rescue of both cilia morphology and ciliary composition. This study, by demonstrating that targeted exon skipping is able to rescue ciliary protein composition defects, provides functional evidence for the efficacy of this approach in the treatment of JBTS.
This book proposes that the UK should develop a federal structure of government with only a small number of functions such as defence and border control being determined at the UK level. All other ...functions would be the ultimate responsibility of individual nations within the UK.
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