28 research outputs found
Spatial Localization of Vitamin D Metabolites in Mouse Kidney by Mass Spectrometry Imaging
Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD-MALDI-MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD-MSI by adapting existing CD methods
Spatial distribution of isobaric androgens in target tissues using chemical derivatization and MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument
Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumour regression, but commonly restricted by the eventual emergence of a more lethal âcastrate resistantâ (CRPC) form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). Decoding this tissue-specific metabolic pathway is key for the development of novel therapeutic treatments. Mass spectrometry imaging (MSI) is an analytical technique that allows the visualization of the distribution of numerous classes of biomolecules within tissue sections. The analysis of androgens by liquid chromatography mass spectrometry (LC/MS)-based methods however presents a challenge due to their generally poor ionization efficiency and low physiological endogenous levels. In MSI, on-tissue chemical derivatization (OTCD) has enabled the limits of steroids to be imaged within tissues to be pushed by overcoming poor ionization performance. However, isobaric interference of key androgen derivatives such as T and DHEA can severely hamper studying the intracrinology in several diseases. Here, we have evaluated the use of laser induced post-ionization (MALDI-2) combined with trapped ion mobility separation (TIMS) and orthogonal time-of-flight (QTOF) MS for the visualization of isobaric derivatized androgens in murine tumour xenograft at about 50 ÎŒm spatial resolution. With this combination, isobaric T and DHEA were separated in tissue sections and the signals of derivatized steroids enhanced by about 20 times. The combination of TIMS and MALDI-2 thus shows unique potential to study tissue intracrinology within target tissues. This could offer the opportunity for many novel insights into tissue-specific androgen biology
Novel nut and bolt task quantifies motor deficits in premanifest and manifest Huntington's Disease
BACKGROUND: We investigated the use of a simple novel nut and bolt task in premanifest and manifest Huntington's disease (HD) patients to detect and quantify motor impairments at all stages of the disease. METHODS: Premanifest HD (n=24), manifest HD (n=27) and control (n=32) participants were asked to screw a nut onto a bolt in one direction, using three different sized bolts with their left and right hand in turn. RESULTS: We identified some impairments at all stages of HD and in the premanifest individuals, deficits in the non-dominant hand correlated with disease burden scores. CONCLUSION: This simple, cheap motor task was able to detect motor impairments in both premanifest and manifest HD and as such might be a useful quantifiable measure of motor function for use in clinical studies
On-Tissue Chemical Derivatization in Mass Spectrometry Imaging
Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrixâassisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed onâtissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCDâMSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive
Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment
Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryoâelectron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications
Evaluation of a COVID â19 fundamental nursing care guideline versus usual care: The COVIDâNURSE cluster randomized controlled trial
Aim: To evaluate the impact of usual care plus a fundamental nursing care guideline compared to usual care only for patients in hospital with COVIDâ19 on patient experience, care quality, functional ability, treatment outcomes, nurses' moral distress, patient healthârelated quality of life and costâeffectiveness. Design: Parallel twoâarm, clusterâlevel randomized controlled trial. Methods: Between 18th January and 20th December 2021, we recruited (i) adults aged 18 years and over with COVIDâ19, excluding those invasively ventilated, admitted for at least three days or nights in UK Hospital Trusts; (ii) nurses caring for them. We randomly assigned hospitals to use a fundamental nursing care guideline and usual care or usual care only. Our patientâreported coâprimary outcomes were the Relational Aspects of Care Questionnaire and four scales from the Quality from the Patient Perspective Questionnaire. We undertook intentionâtoâtreat analyses. Results: We randomized 15 clusters and recruited 581 patient and 418 nurse participants. Primary outcome data were available for 570â572 (98.1%â98.5%) patient participants in 14 clusters. We found no evidence of betweenâgroup differences on any patient, nurse or economic outcomes. We found betweenâgroup differences over time, in favour of the intervention, for three of our five coâprimary outcomes, and a significant interaction on one primary patient outcome for ethnicity (white British vs. other) and allocated group in favour of the intervention for the âotherâ ethnicity subgroup. Conclusion: We did not detect an overall difference in patient experience for a fundamental nursing care guideline compared to usual care. We have indications the guideline may have aided sustaining good practice over time and had a more positive impact on nonâwhite British patients' experience of care. Implications for the Profession and/or Patient Care: We cannot recommend the wholescale implementation of our guideline into routine nursing practice. Further intervention development, feasibility, pilot and evaluation studies are required. Impact: Fundamental nursing care drives patient experience but is severely impacted in pandemics. Our guideline was not superior to usual care, albeit it may sustain good practice and have a positive impact on nonâwhite British patients' experience of care. Reporting Method: CONSORT and CONSERVE. Patient or Public Contribution: Patients with experience of hospitalization with COVIDâ19 were involved in guideline development and writing, trial management and interpretation of findings
Application of an affinity chromatography toolbox to drug repurposing for cancer therapeutics
Phenotypic screening of drug molecules relies on the generation of a specific response;
however the means by which this is elicited often remains unknown. Affinity
chromatography is a valuable tool in the discovery of drug binding partners and may
even allow the elucidation of the wider interactome of the initial drug target. The
introduction of easily cleavable linkers and affinity-independent elution protocols to
affinity chromatography is of current interest, since they render the technique much
more adaptable with respect to the characterisation of biologically active species of
interest. This thesis details the application of a novel azobenzene linker developed by
the Hulme group for use in affinity-independent chromatography.
The first chapter reviews recent developments in affinity chromatography and
describes the synthesis of an affinity linker toolbox with both affinity-dependent and
affinity-independent linkers. These linkers are functionalised with an azide moiety for
use in CuAAC coupling to alkynyl derivatives of bioactive small molecules and have
been modified to include photoreactive groups giving a series of linkers for use in the
identification of less abundant, or low affinity, proteins.
The first drug investigated, anisomycin (ANS), is a small molecule which was initially
introduced as an antibiotic drug (Flagecidin). At nanomolar concentrations ANS has
been shown to affect the mitogen activated protein kinase (MAPK) pathways;
downstream effects of these pathways are thought to play a role in a range of
pathological disorders such as Alzheimerâs disease, cancer and spinal muscular
atrophy (SMA). ANS is thus a candidate for drug repurposing. Although the
downstream effects of MAPK/SAPK pathway activation induced by anisomycin are
well-documented, the cellular target has yet to be revealed. Previous work by the
Hulme group has shown that the N-propargyl anisomycin derivative (I) retains the
biological activity of the lead compound ANS. Thus to evaluate the cellular protein
targets, N-propargyl ANS (I) was coupled onto the linker toolbox to create an ANS
affinity probe library as described in chapter 2.
The second drug investigated, fingolimod, was introduced as an immunomodulating
drug (Glienya) for the treatment of multiple sclerosis (MS). This small molecule has
also been shown to have anti-cancer properties in a range of cancer cell lines; however
the precise mechanism by which this is effected is unknown. Literature precedent
shows that terminal modification of fingolimod generates analogues which still retain
biological activity. Thus a novel fingolimod alkyne derivative (II) was synthesised
and used to create an affinity probe library as described in chapter 3.
Chapter 4 describes affinity pull-down experiments conducted with the aim of finding
the protein target(s) of ANS and fingolimod, using the affinity probe libraries
generated in chapters 2 and 3. This chapter concludes with a discussion of the
implications of these findings and directions for future study