An isotopically-labelled temporal mass spectrometry imaging data analysis workflow to reveal glucose spatial metabolism patterns in bovine lens tissue.

Application of stable isotopically labelled (SIL) molecules in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) over a series of time points allows the temporal and spatial dynamics of biochemical reactions to be tracked in a biological system. However, these large kinetic MSI datasets and the inherent variability of biological replicates presents significant challenges to the rapid analysis of the data. In addition, manual annotation of downstream SIL metabolites involves human input to carefully analyse the data based on prior knowledge and personal expertise. To overcome these challenges to the analysis of spatiotemporal MALDI-MSI data and improve the efficiency of SIL metabolite identification, a bioinformatics pipeline has been developed and demonstrated by analysing normal bovine lens glucose metabolism as a model system. The pipeline consists of spatial alignment to mitigate the impact of sample variability and ensure spatial comparability of the temporal data, dimensionality reduction to rapidly map regional metabolic distinctions within the tissue, and metabolite annotation coupled with pathway enrichment modules to summarise and display the metabolic pathways induced by the treatment. This pipeline will be valuable for the spatial metabolomics community to analyse kinetic MALDI-MSI datasets, enabling rapid characterisation of spatio-temporal metabolic patterns from tissues of interest.

Effects of protein variations by different extraction and dehydration approaches on hempseed protein isolate: Protein pattern, amino acid profiling and label-free proteomics.

This study evaluates the effects of alkaline and micellisation extraction methods, alongside freeze-drying and spray-drying, on the protein subunits, amino acid profiles, and proteome data of hempseed protein isolate (HPI). Findings revealed that the extraction methods affect protein profiles more than the drying methods. Micellisation-extracted HPI showed higher albumin, oleosin, and sulphur-containing protein levels than alkaline-extracted HPI. The alkali-extracted undried sample (AU) gave more potentially allergenic proteins, including Hsp70 and triosephosphate isomerase, than its micellization-extracted counterpart (MU). Unique potential allergens were identified, including malate dehydrogenase and enolase in AU, and RuBisCo in MU samples. Both drying processes impacted the HPI proteome and reduced RuBisCo in the micellisation-extracted HPI. These insights highlight the crucial role of method selection in HPI processing for optimising production in the food industry.

The tumour-derived extracellular vesicle proteome varies by endometrial cancer histology and is confounded by an obesogenic environment.

Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0 µg protein: 1.38 × 1011-1.10 × 1012 particles), characterised by their size (∼150 nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2).

Mapping Glucose Uptake, Transport and Metabolism in the Bovine Lens Cortex.

Purpose: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions. Methods: Ex vivo bovine lenses were incubated in artificial aqueous humour containing normoglycaemic stable isotopically-labelled (SIL) glucose (5 mM) for 5 min-20 h. Following incubations, lenses were frozen for subsequent matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) analysis using high resolution mass spectrometry. Manually dissected, SIL-incubated lenses were subjected to gas chromatography-mass spectrometry (GC-MS) to verify the identity of metabolites detected by MALDI-IMS. Normal, unincubated lenses were manually dissected into epithelium flat mounts and fibre cell fractions and then subjected to either gel-based proteomic analysis (Gel-LC/MS) to detect facilitative glucose transporters (GLUTs) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Indirect immunofluorescence and confocal microscopy of axial lens sections from unincubated fixed lenses labelled with primary antibodies specific for GLUT 1 or GLUT 3 were utilised for protein localisation. Results: SIL glucose uptake at 5 min was concentrated in the equatorial region of the lens. At later timepoints, glucose gradually distributed throughout the epithelium and the cortical lens fibres, and eventually the deeper lens nucleus. SIL glucose metabolites found in glycolysis, the sorbitol pathway, the pentose phosphate pathway, and UDP-glucose formation were mapped to specific lens regions, with distinct regional signal changes up to 20 h of incubation. Spatial proteomic analysis of the lens epithelium detected GLUT1 and GLUT3. GLUT3 was in higher abundance than GLUT1 throughout the epithelium, while GLUT1 was more abundant in lens fibre cells. Immunohistochemical mapping localised GLUT1 to epithelial and cortical fibre cell membranes. Conclusion: The major uptake site of glucose in the bovine lens has been mapped to the lens equator. SIL glucose is rapidly metabolised in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres, with low levels of metabolic activity observed in the nucleus.

Resolving the developmental distribution patterns of polyphenols and related primary metabolites in bilberry (Vaccinium myrtillus) fruit.

Bilberry (Vaccinium myrtillus) is a commercially important wild berry species, which accumulates high amounts of polyphenols, particularly anthocyanins, in the skin and flesh. Whilst a number of studies have quantified these phytochemicals in intact ripe bilberry fruit, we extend the current knowledge by investigating the spatial distribution of anthocyanin-associated polyphenols in fruit tissue, and study their links with primary metabolism during ripening. To address this, we used LC-MS and mass spectrometry imaging to measure and map primary and secondary metabolites in fruit. Correlation analysis showed that five sugars displayed strong positive correlations with anthocyanin accumulation, whereas all amino acids were negatively correlated. The accumulation patterns of polyphenols correlated in fruit skin and flesh, but altered with development. Finally, spatial segmentation analysis revealed that the chemical signatures of ripening first appear at defined regions under the skin and rapidly expand to encompass the entire fruit at the eating-ripe stage.

Investigating the consistency of extracellular vesicle production from breast cancer subtypes using CELLine adherent bioreactors.

Extracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in-depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV-associated proteins are relatively consistent, with few minor variations over time, but that tracking the production of EVs is a convenient method to indirectly monitor the bioreactor and consistency of the yielded EVs. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.

Free-Standing N-Doped Porous Carbon Fiber Membrane Derived From Zn-MOF-74: Synthesis and Application as Anode for Sodium-Ion Battery With an Excellent Performance.

It is important to develop new energy storage and conversion technology to mitigate the energy crisis for the sustainable development of human society. In this study, free-standing porous nitrogen-doped carbon fiber (PN-CF) membranes were obtained from the pyrolysis of Zn-MOF-74/polyacrylonitrile (PAN) composite fibers, which were fabricated in situ by an electrospinning technology. The resulting free-standing fibers can be cut into membrane disks and directly used as an anode electrode without the addition of any binder or additive. The PN-CFs showed great reversible capacities of 210 mAh g-1 at a current density of 0.05 A g-1 and excellent cyclic stability of 170.5 mAh g-1 at a current density of 0.2 A g-1 after 600 cycles in sodium ion batteries (SIBs). The improved electrochemical performance of PN-CFs can be attributed to the rich porous structure derived by the incorporation of Zn-MOF-74 and nitrogen doping to promote sodium ion transportation.

Mass Spectrometry-Based Metabolomics to Investigate the Effect of Mechanical Shaking on Sauvignon Blanc Berry Metabolism.

Previous commercial studies carried out in New Zealand showed that mechanical shaking significantly reduced the incidence of Botrytis cinerea infection in wine grapes. However, the reasons behind this reduction are not well understood. Here, we employed a metabolomics approach to gain insights into the biochemical changes that occur in grape berries due to mechanical shaking. Berry samples were analyzed using three different analytical approaches including gas chromatography and mass spectrometry (MS), liquid chromatography and MS, and imaging mass spectrometry (IMS). Combined data provided a comprehensive overview of metabolic changes in grape berry, indicating the initiation of different stress mitigation strategies to overcome the effect of mechanical shaking. Berry primary metabolism was distinctly altered in the green berries in response to mechanical shaking, while secondary metabolism significantly changed in berries collected after veraison. Pathway analysis showed upregulation of metabolites related to nitrogen and lipid metabolism in the berries from shaken vines when compared with controls. From IMS data, we observed an accumulation of different groups of metabolites including phenolic compounds and amino and fatty acids in the areas near to the skin of berries from shaken vines. This observation suggests that mechanical shaking caused an accumulation of these metabolites, which may be associated with the formation of a protective barrier, leading to the reduction in B. cinerea infection in berries from mechanically shaken vines.

Applications of stable isotopes in MALDI imaging: current approaches and an eye on the future.

Matrix-assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) is now an established imaging modality with particular utility in the study of biological, biomedical and pathological processes. In the first instance, the use of stable isotopically labelled (SIL) compounds in MALDI-IMS has addressed technical barriers to increase the accuracy and versatility of this technique. This has undoubtedly enhanced our ability to interpret the two-dimensional ion intensity distributions produced from biological tissue sections. Furthermore, studies using delivery of SIL compounds to live tissues have begun to decipher cell, tissue and inter-tissue metabolism while maintaining spatial resolution. Here, we review both the technical and biological applications of SIL compounds in MALDI-IMS, before using the uptake and metabolism of glucose in bovine ocular lens tissue to illustrate the current limitations of SIL compound use in MALDI-IMS. Finally, we highlight recent instrumentation advances that may further enhance our ability to use SIL compounds in MALDI-IMS to understand biological and pathological processes. Graphical Abstract.

Mapping glucose metabolites in the normal bovine lens: Evaluation and optimisation of a matrix-assisted laser desorption/ionisation imaging mass spectrometry method.

The spatial resolution of microdissection-based analytical methods to detect ocular lens glucose uptake, transport and metabolism are poor, whereas the multiplexing capability of fluorescence microscopy-based approaches to simultaneously detect multiple glucose metabolites is limited in comparison with mass spectrometry-based methods. To better understand lens glucose transport and metabolism, a more highly spatially resolved technique that maintains the fragile ocular lens tissue is required. In this study, a sample preparation method for matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) analysis of ocular lens glucose uptake and metabolism has been evaluated and optimised. Matrix choice, tissue preparation and normalisation strategy were determined using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS of bovine lens tissue and validation performed using gas chromatography-MS. An internal standard was applied concurrently with N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC) matrix to limit cracking of the fresh frozen lens tissue sections. MALDI IMS data were collected at a variety of spatial resolutions to detect both endogenous lens metabolites and stable isotopically labelled glucose introduced by ex vivo lens culture. Using this approach, initial steps in important metabolic processes that are linked to diabetic cataract formation were spatially mapped in the bovine lens. In the future, this method can be applied to study the dynamics of glucose uptake, transport and metabolic flux to aid in the study of diabetic lens cataract pathophysiology.

Tablet-Based Patient-Centered Decision Support for Minor Head Injury in the Emergency Department: Pilot Study.

BACKGROUND: The Concussion or Brain Bleed app is a clinician- and patient-facing electronic tool to guide decisions about head computed tomography (CT) use in patients presenting to the emergency department (ED) with minor head injury. This app integrates a patient decision aid and clinical decision support (using the Canadian CT Head Rule, CCHR) at the bedside on a tablet computer to promote conversations around individualized risk and patients' specific concerns within the ED context. OBJECTIVE: The objective of this study was to describe the use of the Concussion or Brain Bleed app in a high-volume ED and to establish preliminary efficacy estimates on patient experience, clinician experience, health care utilization, and patient safety. These data will guide the planning of a larger multicenter trial testing the effectiveness of the Concussion or Brain Bleed app. METHODS: We conducted a prospective pilot study of adult (age 18-65 years) patients presenting to the ED after minor head injury who were identified by participating clinicians as low risk by the CCHR. The primary outcome was patient knowledge regarding the injury, risks, and CT use. Secondary outcomes included patient satisfaction, decisional conflict, trust in physician, clinician acceptability, system usability, Net Promoter scores, head CT rate, and patient safety at 7 days. RESULTS: We enrolled 41 patients cared for by 29 different clinicians. Patient knowledge increased after the use of the app (questions correct out of 9: pre-encounter, 3.3 vs postencounter, 4.7; mean difference 1.4, 95% CI 0.8-2.0). Patients reported a mean of 11.7 (SD 13.5) on the Decisional Conflict Scale and 92.5 (SD 12.0) in the Trust in Physician Scale (both scales range from 0 to 100). Most patients were satisfied with the app's clarity of information (35, 85%), helpfulness of information (36, 88%), and amount of information (36, 88%). In the 41 encounters, most clinicians thought the information was somewhat or extremely helpful to the patient (35, 85%), would want to use something similar for other decisions (27, 66%), and would recommend the app to other providers (28, 68%). Clinicians reported a mean system usability score of 85.1 (SD 15; scale from 0 to 100 with 85 in the "excellent" acceptability range). The total Net Promoter Score was 36.6 (on a scale from -100 to 100). A total of 7 (17%) patients received a head CT in the ED. No patients had a missed clinically important brain injury at 7 days. CONCLUSIONS: An app to help patients assess the utility of CT imaging after head injury in the ED increased patient knowledge. Nearly all clinicians reported the app to be helpful to patients. The high degree of patient satisfaction, clinician acceptability, and system usability support rigorous testing of the app in a larger multicenter trial.

Patient-Centered Decision Support: Formative Usability Evaluation of Integrated Clinical Decision Support With a Patient Decision Aid for Minor Head Injury in the Emergency Department.

BACKGROUND: The Canadian Computed Tomography (CT) Head Rule, a clinical decision rule designed to safely reduce imaging in minor head injury, has been rigorously validated and implemented, and yet expected decreases in CT were unsuccessful. Recent work has identified empathic care as a key component in decreasing CT overuse. Health information technology can hinder the clinician-patient relationship. Patient-centered decision tools to support the clinician-patient relationship are needed to promote evidence-based decisions. OBJECTIVE: Our objective is to formatively evaluate an electronic tool that not only helps clinicians at the bedside to determine the need for CT use based on the Canadian CT Head Rule but also promotes evidence-based conversations between patients and clinicians regarding patient-specific risk and patients' specific concerns. METHODS: User-centered design with practice-based and participatory decision aid development was used to design, develop, and evaluate patient-centered decision support regarding CT use in minor head injury in the emergency department. User experience and user interface (UX/UI) development involved successive iterations with incremental refinement in 4 phases: (1) initial prototype development, (2) usability assessment, (3) field testing, and (4) beta testing. This qualitative approach involved input from patients, emergency care clinicians, health services researchers, designers, and clinical informaticists at every stage. RESULTS: The Concussion or Brain Bleed app is the product of 16 successive iterative revisions in accordance with UX/UI industry design standards. This useful and usable final product integrates clinical decision support with a patient decision aid. It promotes shared use by emergency clinicians and patients at the point of care within the emergency department context. This tablet computer app facilitates evidence-based conversations regarding CT in minor head injury. It is adaptable to individual clinician practice styles. The resultant tool includes a patient injury evaluator based on the Canadian CT Head Rule and provides patient specific risks using pictographs with natural frequencies and cues for discussion about patient concerns. CONCLUSIONS: This tool was designed to align evidence-based practices about CT in minor head injury patients. It establishes trust, empowers active participation, and addresses patient concerns and uncertainty about their condition. We hypothesize that, when implemented, the Concussion or Brain Bleed app will support-not hinder-the clinician-patient relationship, safely reduce CT use, and improve the patient experience of care.

Calcitonin gene-related peptide is a promising marker in ulcerative colitis.

BACKGROUND: Assessment of the severity and extent of disease activity continues to present challenges for physicians in the treatment of ulcerative colitis. Standard markers that can objectively reflect disease activity are useful for physicians to both evaluate the course of ulcerative colitis and monitor the effectiveness of therapy for any given patient. AIMS: We hypothesize that calcitonin gene-related peptide (CGRP) can reflect the activity and severity of ulcerative colitis and be used as a marker to assess the effectiveness of various therapies. METHODS: We examined the expression levels of CGRP by reverse transcription polymerase chain reaction (RT-PCR) and semi-quantitative immunohistochemisty in mucosal biopsies from 38 patients with UC and 18 controls. Levels of CGRP mRNA and protein expression were compared between patients and controls with the clinical activity index (CAI) and the endoscopic activity index (EAI) for various levels of UC severity. RESULTS: Our results showed that the levels of CGRP mRNA and protein expression were significantly reduced in UC patients compared to controls. This effect was more pronounced in patients with more severe cases of UC. There is a statistically significant negative correlation between levels of CGRP mRNA expression and CAI/EAI scores. A statistically significant negative correlation was also found between levels of CGRP protein expression and CAI/EAI scores. Overall, high CAI and EAI scores were accompanied by low CGRP mRNA and protein expression levels. CONCLUSION: Levels of CGRP protein and mRNA expression in the colonic mucosa of patients are closely associated with UC severity and corroborate traditional indices used to assess the disease.

Laser-guided cell micropatterning system.

Employing optical force, our laser-guided cell micropatterning system, is capable of patterning different cell types onto and within standard cell research devices, including commercially available multielectrode arrays (MEAs) with glass culture rings, 35 mm Petri dishes, and microdevices microfabricated with polydimethylsiloxane on 22 mm × 22 mm cover glasses. We discuss the theory of optical forces for generating laser guidance and the calculation of optimal beam characteristics for cell guidance. We describe the hardware design and software program for the cell patterning system. Finally, we demonstrate the capabilities of the system by (1) patterning neurons to form an arbitrary pattern, (2) patterning neurons onto the electrodes of a standard MEA, and (3) patterning and aligning adult cardiomyocytes in a polystyrene Petri dish.

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid.

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.