Analytical Biochemistry

Coenzyme A is an essential cofactor synthesized from pantothenate, cysteine, and ATP, and is involved in numerous processes of cellular metabolism through its ability to carry activated acyl groups. Coenzyme A participates in catabolism of carbohydrate, fat and amino acids; biosynthesis of fatty acids, cholesterol and heme; and protein modification including acetylation and 4-phosphopantetheinylation. Despite CoAs critical functions, the regulation of CoA levels and the rate of CoA synthesis in different cell types and disease states are not well understood. One reason for this gap is that many acyl-CoA species are analytically challenging to measure due to factors including instability, poor ionization, and the wide range of biochemical properties conferred by different acyl chain lengths. In addition, most current methods do not support analysis of CoA isotopic labeling, which is required to quantify CoA synthesis rate or to measure absolute concentration using isotope-labeled internal standards. Here, we describe a method to quantify the concentration and isotopic labeling of total CoA, defined as the sum of CoASH plus all acyl-CoA species. Acyl-CoA species are hydrolyzed using sodium hydroxide to remove acyl chains, then CoA is derivatized on the thiol with N-ethylmaleimide (NEM). Following protein precipitation and solid phase extraction, samples are analyzed by liquid chromatography-mass spectrometry. This method is linear in a wide range that captures mouse tissue CoA levels, with accuracy within 15% error and precision below 15% relative standard deviation for both pure standards and tissue samples. We applied this method to measure total CoA concentration in five tissues from male and female mice, and total CoA synthesis rate in mouse liver via infusion of 13C-15N-pantothenate. Overall, this method offers a tractable approach to measure total CoA concentration and isotopic labeling to enable study of total CoA synthesis rates and concentrations in health and disease.

1Sex steroid hormones are not exclusively localised in the circulation and can be found in numerous extragonadal tissues, in concentrations unrelated to the circulating fraction. Existing methodology to measure intramuscular steroid hormone concentrations includes both immune-based assays and liquid chromatography-mass spectrometry (LC-MS), the gold standard for hormone measurements. To date, no LC-MS based methods validation has been published on the measurement of intramuscular sex steroid hormones, despite clear biological relevance. Here, we describe the development and validation of a simple, high-throughput LC-MS Orbitrap method for the measurement of 10 intramuscular sex steroid hormones, including pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, epitestosterone, dihydrotestosterone, oestrone, oestradiol, and oestriol. In brief, isotope labelled standards were added to 5-6 milligrams of lyophilised muscle tissue, homogenised and extracted with ethyl acetate. The extracts were dried down and sequentially derivatised with 1-methylimidazole-2-sulfonyl chloride and hydroxylamine hydrochloride to target both the phenolic hydroxyl groups and ketone groups. The limit of detection was 1.0 {+/-} 1.0 pg/mg (range 0.36 - 3.26 pg/mg), with a R2 > 0.99 for all analytes. Matrix effects were 90-110% for all analytes except for dihydrotestosterone (143.6%), and precision was <10 CV% for all analytes in the presence of a muscle matrix. Our method allows for 20-40 samples to be prepared in [~]4 h, with a sample data acquisition time of 13 minutes. Moreover, our method provides the opportunity for specific analysis of steroid hormone concentrations in skeletal muscle, allowing target tissue specificity instead of relying on proxy measures from the circulation.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

Authentication of acai products is increasingly important due to the risk of species substitution among morphologically similar Euterpe taxa, with implications for food quality, labeling accuracy, and consumer trust. Despite advances in molecular methods, rapid and cost-effective tools for discriminating closely related Euterpe species in processed commercial matrices remain limited. This study evaluated High-Resolution Melting (HRM) analysis targeting two complementary chloroplast markers -- psbK-I and ycf1b -- as a practical approach for species-level authentication of acai (Euterpe oleracea and E. precatoria) and jucara (E. edulis) products. In silico specificity analysis confirmed that the ycf1b primer pair shows amplification restricted to the Arecaceae family, supporting the analytical robustness of the method. The combined markers enabled reliable differentiation of all target species, including closely related taxa, with a detection limit of approximately 10% in admixed samples. When applied to 50 commercial products, HRM successfully authenticated 46 samples, substantially outperforming DNA sequencing, which was limited by amplification failure and mixed chromatograms. Mislabeling was detected in one acai sorbet and three frozen acai pulps marketed as acai but molecularly identified as E. edulis, constituting a violation of Brazilian food labeling regulations. These findings demonstrate that HRM analysis provides a robust, rapid, and scalable strategy for routine species authentication in processed plant-based matrices, with potential for integration into food quality control workflows and large-scale commercial monitoring programs.

The protein sample preparation methods for shotgun proteomics are nowadays well-established unlike the ones for whole protein analysis. The goal of my work has been to create a simple methodology which provides a single uncomplicated sample preparation tool for these two fields. Nowadays the bulk of proteomics work is done using detergents for protein solubilization. The presented concept, which is based on unspecific adsorption of protein molecules on wide pore materials, allows for protein capture and clean-up from solutions of the most commonly used sodium dodecyl sulfate detergent. It could also be applied to proteins in detergent-free solutions. After the capture and clean-up, proteins could be either cleaved for the downstream peptide analysis or eluted for the whole protein analysis. If required, the eluted whole proteins could be recaptured and cleaved into peptides. Depending on the experimental goals, the sample preparation device could be fitted with embedded proteolytic enzymes to simplify routine sample processing and/or reversed phase media for the downstream peptide or protein separation.

Phytohormones are key players in the regulation of plant development and metabolism. The different phytohormone classes comprise numerous chemically very diverse compounds, which are often present at very low concentrations. The chemical properties of phytohormones range from acidic to basic and from polar to non-polar. Furthermore, concentration varies strongly among different phytohormones, between plant species, tissues and developmental stages. Challenges often arise when only small amounts of plant material are available and when plant species are investigated in which the phytohormone profile has not yet been characterized. To establish a method for comprehensive phytohormone analysis we addressed these challenges by choosing and optimizing a suitable extraction method followed by optimized HPLC separation. We compared the most widely-used mass spectrometric detection methods, multiple reaction monitoring (MRM) on a triple quad instrument with high-resolution mass spectrometry (HRMS) on a Q-TOF instrument, and discuss the advantages of both methods and their limitations. O_LIWe compared various methods described in literature for the extraction of six phytohormone classes by liquid-liquid extraction and solid phase extraction purification and describe our optimizations to the selected method. C_LIO_LIWe optimized HPLC separation for 50 different phytohormones. C_LIO_LIWe evaluated the application of MRM and HRMS detection strategies. C_LI

Lipid metabolism is increasingly recognized as a hallmark of cancer, yet translating lipidomic discoveries into clinically actionable biomarkers remains constrained by analytical variability and limited standardized validation frameworks. This challenge is further compounded by a chicken-or-egg problem, where expensive standards and labelled internal standards are required to identify and quantitate target lipids, but the diagnostic importance of these targets is uncertain until they can be reliably measured. Previous work had indicated the potential of 48 lipid biomarker species for the prediction of breast cancer from plasma samples using high resolution liquid chromatography mass spectrometry. This study aimed to identify each of these 48 species and develop a quantitative method to determine the absolute concentrations of these lipids in plasma to provide the basis for the development of a clinical assay for use in breast cancer detection. In doing so, we present a pragmatic workflow that bridges lipid discovery with lipid identification and robust quantitative analysis. A curated library of 48 lipid species was established using authentic standards to verify plasma lipids through retention-time matching and high-resolution spectral comparison. In plasma, 41 lipids were confidently identified based on co-elution with standards and diagnostic fragment ions. Method qualification, including assessment of accuracy, precision, recovery, and linearity, was performed across all 48 lipids in parallel with identification, and 46 lipids ultimately met all predefined qualification criteria. Notably, practical constraints, including time, cost, and availability of authentic standards, necessitated performing identification and targeted method development in parallel, highlighting challenges inherent to translating lipidomics into commercial or clinical assays. This workflow provides a reproducible framework for harmonizing lipid identification and quantification, enabling the reliable integration of lipidomic data into biomarker discovery and clinical applications.

Human milk contains structurally diverse glycans with key roles in shaping infant development, yet analytical constraints limit characterisation from low-volume samples. Glycosaminoglycans (GAGs), including chondroitin sulphate (CS), are understudied due to existing protocols requiring sample volumes of at least 5 mL and lengthy extraction steps prior to instrumental analysis. This study establishes a workflow for quantifying CS disaccharides from 25 {micro}L of human milk, enabling analysis of samples previously inaccessible to GAG profiling, such as those collected as salvage samples from neonatal intensive care units. For CS quantification, the CS is first enzymatically depolymerised using chondroitinase ABC to release repeating disaccharide units. Matrix complexity is reduced via two rounds of acetonitrile-based protein and lipid precipitation. Disaccharides are separated by hydrophilic interaction liquid chromatography and detected using a Triple Quadrupole Mass Spectrometer, providing robust sensitivity for all CS disaccharides. Method development and validation were performed using pooled mature human milk from term infants. This workflow facilitates detection of all CS disaccharides, with low but reproducible recoveries for total CS. Low- and high-level spike recoveries were 41.3% (RSDr 7.5%, RSDiR 15.9%) and 43.7% (RSDr 24.4%, RSDiR 27.9%), respectively. Despite modest absolute accuracy, precision remained sufficient to make relative comparison of CS concentrations between samples. This method expands the analytical toolkit for human milk glycomics, enabling same day preparation and CS profiling from sample volumes that are 200 times smaller than prior work, supporting future investigations into GAG-mediated functions in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723732v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@176dffborg.highwire.dtl.DTLVardef@16ae4ccorg.highwire.dtl.DTLVardef@d333c2org.highwire.dtl.DTLVardef@1eb3216_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Schematic of sample preparation protocol 25 L of human milk is combined with lyase enzymes and TRIS buffer containing the internal standard prior to incubation. Samples then undergo multiple rounds of centrifugation and refrigeration before analysis via LC-MS/MS. Made using BioRender.com. Glycan nomenclature following Varki et al., 2015. C_FIG

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in immunopeptidomics, structural proteomics, and analyses of post-translational modifications. In such analyses, peptides that are not biologically meaningful but instead arise as artifacts prior to mass spectrometry analysis pose the risk of data misinterpretation. Here, we describe an approach based on retention time analysis and precise chromatographic peak matching to identify peptides generated by in-source fragmentation (ISF), which occurs between chromatographic separation of peptide mixtures and the first mass filter of a tandem mass spectrometer (MS). To understand the prevalence and properties of ISF, we generated 13 proteomics datasets and analyzed them along with additional 25 previously published datasets spanning a broad range of sample types, MS, and proteomics approaches including classical bottom-up proteomics, immunopeptidomics, structural proteomics, and phosphoproteomics. We found that, in typical trypsin-digested samples on average 1 % of fully-tryptic peptides and 22 % of semi-tryptic peptides originated from ISF. However, we observed large variations between datasets, and in-source fragments exceeded, in some cases, a third of the total peptide identifications. The extent of ISF was dependent on the peptide sequence, the instrument, method parameters, and sample complexity. Although ISF did not impair relative quantification across samples, it generated peptides that could be misinterpreted qualitatively, inflated peptide identifications, and comprised up to 37 percent of peptides shorter than 9 amino acids in immunopeptidomics datasets. We propose that, for peptide-centric applications, our open-source ISF detection approach be used to re-annotate peptides generated by ISF and remove them to avoid misinterpretation of data. ISF is an increasing concern with improving mass spectrometers, as they enable detection of an ever-increasing number of m/z features, including low abundance features like ISF products. Our work thus addresses a growing issue in proteomics and presents solutions to mitigate the impact of in-source fragment peptides. In the future, improved feature detection algorithms may enable elucidation of new ISF patterns affecting side chains that have been missed so far, which could contribute to explaining the vast space of as-yet unannotated proteomics data.

BackgroundDynamic contrast-enhanced (DCE) MRI has the potential to be a useful tool for non-invasively assessing renal haemodynamics and function, however insufficient standardisation and difficulties in post-processing remain barriers to clinical translation. PurposeTo develop expert consensus-based technical recommendations for performing renal DCE-MRI in humans, relating to aspects of patient preparation, MRI hardware and acquisition parameters, and data analysis. Study TypeSystematic consensus process using an approximation to the two-step modified Delphi method. PopulationNot applicable. Field Strength / Sequence1.5 T and 3 T / Renal gradient echo-based 3D DCE-MRI. AssessmentAn international panel of experts were recruited and surveyed following a modified Delphi method to create consensus-based technical recommendations. Key areas for consensus were initially identified through a mixture of online and in-person discussions, and an initial survey round consisting of open- and close-ended questions. Consensus statements were formulated and iteratively refined to create the final recommendations. Statistical TestsConsensus was defined as [&ge;] 75% agreement in response (excluding abstentions), and clear preference was defined as [60-74]% agreement among the experts. Statements with [&ge;]40% abstentions were either excluded from subsequent survey rounds or recirculated as a modified statement. Results22 experts initially participated in the Delphi panel, of which 16 responded to the first survey. 15 panellists responded to all subsequent surveys. Out of 46 statements, 37 reached consensus and one showed clear preference. [&ge;]40% abstention was found in seven statements which were excluded from the final set of recommendations. Data conclusionThese recommendations provide a starting point for MRI centres worldwide wishing to perform renal DCE-MRI, contributing to the harmonisation of DCE-MRI scan protocols and facilitating clinical translation. These recommendations provide a practical minimum technical dataset for renal DCE-MRI acquisition and analysis to improve cross-site comparability and support responsible clinical translation.

Magic-angle spinning (MAS) solid-state NMR (SSNMR) has been widely used to determine amyloid fibril structures at atomic resolution. Such studies typically rely on homogeneous fibril preparations that produce narrow linewidths and high spectral resolution, enabling reliable resonance assignment and structural analysis. However, many biologically relevant amyloid aggregates are structurally heterogeneous, resulting in spectral broadening and reduced sensitivity that hinder atomic-resolution characterization. Lipids are known to modulate amyloid aggregation pathways and promote the formation of toxic species that are often less homogeneous, further complicating NMR-based investigations. Here, we evaluate the feasibility of utilizing the benefits associated with high-field (1.1 GHz) SSNMR for studying ganglioside GD3-catalyzed A{beta}42 aggregates. Uniformly-13C,15N-labeled A{beta}42 was incubated with GD3 to generate lipid-associated aggregates and analyzed under MAS conditions. 13C cross-polarization magic-angle spinning (CPMAS) spectra and 2D 13C-13C chemical shift correlation experiments using CORD (COmbined R2nv-Driven) mixing were acquired and compared with data collected at 600 MHz. Despite the heterogeneous nature of the GM1-associated assemblies, the 1.1 GHz spectra exhibit enhanced sensitivity and improved spectral resolution. Better resolved resonances corresponding to selectively structured regions of A{beta}42 are observed, indicating the presence of an ordered core within the lipid-associated aggregates. These results demonstrate that ultrahigh-field SSNMR significantly improves the characterization of heterogeneous amyloid assemblies and provides a promising approach for atomic-level investigation of biologically relevant, lipid-modulated A{beta} aggregates.

BackgroundDiffusion-weighted MRI is central to prostate cancer detection, but apparent diffusion coefficient (ADC) has limited reproducibility across scanners and sites. Restriction Spectrum Imaging restriction score maximum value (RSIrs-max) may provide a more reproducible biomarker. PurposeTo evaluate cross-session reproducibility of within-lesion mean ADC and RSIrs-max on prostate MRI, including same-vendor and cross-vendor comparisons, and in unfavorable-histology prostate cancer (uhPC) and different interpolation settings. Materials and MethodsIn this prospective study, participants with suspected or known prostate cancer enrolled from August 2022 to January 2026 underwent two MRI examinations including an RSI protocol. MRI-visible lesions were contoured on T2-weighted MRI; in participants with multiple lesions, the index lesion was selected. Mean ADC and RSIrs-max were measured within MRI-visible lesions. Analyses included all visible lesions, same-vendor and cross-vendor subgroups, participants with uhPC, and 20 participants with scans reconstructed with and without zero-filled interpolation (a setting with different defaults across vendors). Pearson correlation coefficients with 10,000 bootstrap resamples were used to estimate 95% confidence intervals. ResultsSixty-one male participants (median age, 69 years [IQR, 63-74]) were evaluated; 58 of 61 (95%) had MRI-visible lesions, and 26 of 58 (45%) had uhPC. For all MRI-visible lesions, correlations were 0.55 (95% CI: 0.23-0.76) for mean ADC and 0.83 (95% CI: 0.72-0.90) for RSIrs-max. In same-vendor scans, correlations were 0.76 (95% CI: 0.27-0.95) and 0.88 (95% CI: 0.72-0.96); in cross-vendor scans, they were 0.31 (95% CI: -0.07-0.62) and 0.79 (95% CI: 0.65-0.89), respectively. In uhPC, correlations were 0.42 (95% CI: -0.02-0.83) for mean ADC and 0.90 (95% CI: 0.77-0.96) for RSIrs-max. With inconsistent versus consistent interpolation, RSIrs-max correlation increased from 0.73 (95% CI: 0.48-0.89) to 0.89 (95% CI: 0.78-0.96). ConclusionADC showed limited reproducibility, particularly across vendors. RSIrs-max has stronger between-session reproducibility across same-vendor, cross-vendor, uhPC, and interpolation analyses.

BackgroundMetabolic reprogramming is a hallmark of breast cancer (BrCa), with alterations in glycolysis, glutamine metabolism, and the urea cycle contributing to tumour progression. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, shifts metabolism toward oxidative phosphorylation and has been proposed as a therapeutic agent. While isotope tracing is well-established, natural isotope abundance ({delta}{superscript 1}3C, {delta}{superscript 1}N) is emerging as a biomarker of metabolic alterations in cancer. MethodsWe investigated the relationship between isotope composition and metabolism in BrCa using two BALB/c mouse mammary tumour models (V14 and 4T1) and assessed the effects of DCA treatment using metabolomics, lipidomics and isotopomics. ResultsV14 and 4T1 tumours exhibited isotopic patterns similar to human tumours, with {delta}{superscript 1}3C enrichment and {delta}{superscript 1}N depletion relative to non-cancerous mammary tissue. V14 tumours were more {delta}{superscript 1}N-depleted than 4T1, reflecting differences in nitrogen metabolism. Multivariate analysis integrating isotopic, metabolomic, and lipidomic data revealed isotopic features as key discriminators between tumours and normal tissues. Compared to V14, 4T1 tumours were enriched in TCA intermediates, sphingolipids, and amino acids, whereas V14 tumours showed elevated glutaminolytic and nitrogenous metabolites. DCA treatment differentially affected tumour growth, with V14 tumours more sensitive than 4T1. DCA altered nitrogen metabolism, increasing the arginine-to-ornithine ratio, and modulating {delta}{superscript 1}N values in a tumour-specific manner increasing V14 and decreasing 4T1 {delta}{superscript 1}N values. DCA had little effect on {delta}{superscript 1}3C. {delta}{superscript 1}3C values were primarily determined by the balance between lipid and TCA cycle metabolites, rather than glycolytic flux. {delta}{superscript 1}N variation was linked to nitrogen metabolism, including urea cycle intermediates and sphingolipid composition, with a potential role for choline-related fractionation in {delta}{superscript 1}N depletion. Altered gene expression of Hacd2 and Acot12 in V14 tumours after DCA treatment was reflected in shorter fatty acid tails in phosphatidyl cholines, supporting the lipidomics data. ConclusionsThese findings support the hypothesis that cancer-associated metabolic reprogramming influences natural isotope abundance. Correlations between isotope shifts and metabolic signatures highlight the potential of lipid-derived {delta}{superscript 1}N as a biomarker of tumour metabolic state, with implications for noninvasive metabolic profiling in BrCa. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/710495v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1589d0eorg.highwire.dtl.DTLVardef@af2ad4org.highwire.dtl.DTLVardef@24e67forg.highwire.dtl.DTLVardef@98da7f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Prostate-specific membrane antigen (PSMA) PET/CT is central to prostate cancer staging and theranostic workflows. To our knowledge, no direct within-patient comparison of [18F]FC303 ([18F]Florastamin) and [68Ga]Ga-PSMA-11 has been reported. We performed a preliminary paired method-comparison study under non-harmonized acquisition protocols. Patients and Methods: Twenty patients with histologically confirmed prostate cancer underwent [68Ga]Ga-PSMA-11 PET/CT (185 +/- 37 MBq, 60 +/- 10 min) followed by [18F]FC303 PET/CT (370 +/- 37 MBq, 105 +/- 15 min) on the same PET/CT system within each patient (median interval, 29.5 days). Index targets were anatomically matched to the biopsied or surgically sampled lesion or target region. The primary malignant set included 18 histologically malignant targets; two histology-negative or indeterminate targets were included only in sensitivity analysis. Fixed [68Ga]Ga-PSMA-11-first scan order and the 45-min uptake-time difference were central interpretive constraints. Results: Across five predefined reference organs, [18F]FC303 showed lower SUVmean than [68Ga]Ga-PSMA-11 (all Benjamini-Hochberg-adjusted p < 0.001; [68Ga]/[18F]FC303 geometric mean ratio [GMR], 1.29-3.89). In the primary malignant set, [18F]FC303 lesion SUVmax was lower than [68Ga]Ga-PSMA-11 (median, 11.3 vs 18.1; paired median difference, -5.50; 95% CI, -6.85 to -2.90; Wilcoxon p = 8.4 x 10-4), with strong rank correlation (Spearman {rho} = 0.90). Passing-Bablok regression yielded {beta} = 1.13 (95% CI, 1.04-1.45), and log-Bland-Altman GMR (FC303/[68Ga]) was 0.75, consistent with proportional non-interchangeability. Tumor-to-liver and tumor-to-mediastinum ratios did not differ significantly (GMR, 1.17 [95% CI, 0.94-1.45] and 0.96 [0.80-1.15], respectively); the study was not powered for equivalence. The n = 20 sensitivity analysis showed consistent directionality. Conclusions: Under non-harmonized acquisition conditions, [18F]FC303 showed lower physiologic reference-organ SUVmean and malignant target-region SUVmax than [68Ga]Ga-PSMA-11, whereas tumor-to-liver and tumor-to-mediastinum ratios were not significantly different. Absolute SUVs were not interchangeable; [68Ga]Ga-PSMA-11-derived SUV thresholds should not be directly transferred to [18F]FC303 without tracer-specific calibration.

The extracellular matrix (ECM) and cell surface glycocalyx are key components of biology and play crucial roles in development and tissue function, as well as disease. Proteoglycans, and their glycosaminoglycan (GAG) side chains, are critical components of the ECM and the glycocalyx. GAGs can bind to many different proteins, such as chemokines, and form hydrated barriers around cells. Existing and new methods are helping us to uncover more about the roles of GAGs in biology. Here, we expand on existing technologies and provide streamlined, standardised and well-documented methods that can be easily adopted in standard analytical facilities. We provide extensive detailed step-by-step guides describing sample disruption, GAG disaccharide preparation from biological tissues and their analysis by HILIC-MS/MS. In addition, we demonstrate utility of this method when using a range of different samples as biological sources. This method will sit alongside existing and new techniques to help improve access to GAG analysis, and thereby further the field of understanding GAG function in complex biological contexts.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this study, we demonstrate that urea enables reliable melting temperature (Tm) determination of hyperthermostable proteins by nano differential scanning fluorimetry (nanoDSF) Under native conditions, Pfu DNA polymerase and its Sso7d-fusion variant showed no detectable unfolding transitions, despite their Tm values falling within the instruments operational range, reflecting their extreme kinetic stability. In the presence of up to 7 M urea, intrinsic tyrosine and tryptophan fluorescence revealed clear unfolding transitions, yielding extrapolated Tm values of 104.8 {+/-} 0.09 {degrees}C for Pfu and 106.8 {+/-} 0.33 {degrees}C for its Sso7d-fusion variant. These results demonstrate that urea-gradient nanoDSF overcomes both instrumental and kinetic limitations, providing a simple and robust method for assessing the thermal stability of (hyper)thermostable proteins. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/717478v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@960d9org.highwire.dtl.DTLVardef@1b5613forg.highwire.dtl.DTLVardef@1039a08org.highwire.dtl.DTLVardef@1759841_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mass spectrometry (MS) is a powerful technique for characterizing modified RNA as it directly sequences and quantifies all mass-altering modifications simultaneously. However, the physicochemical properties of RNA result in poor ionization efficiencies during electrospray ionization, presenting a major barrier to sensitive MS measurements necessary for low abundance RNA samples and RNAs with low modification stoichiometries. Here, we report a ligation-based approach to increase ionization efficiencies of RNA oligonucleotides. We show that short ([~]5 nt), chemically modified DNA oligonucleotides can be enzymatically ligated to RNA to serve as MS signal enhancers. Among a series of signal enhancers appended with various alkyl and alkylimidazolium functional groups, we found that decyl-functionalized derivatives improved MS sensitivity by [~]15-fold compared to unlabeled oligonucleotide. When ligated to RNA standards, the decyl-modified signal enhancer increased MS signals 2-4-fold with the additional benefit of improved retention during liquid chromatography (LC) separations without ion pairing agents. To apply the ligation-based approach to RNase T1 digests of longer RNAs, a multi-step enzymatic approach was optimized to maximize ligation efficiencies. We then ligated signal enhancers to a yeast transfer RNA (tRNA) digest and observed increased MS signals for numerous sequence-informative digestion products. Importantly, the sequences of RNA oligonucleotides ligated to signal enhancers were readily determined by tandem mass spectrometry with collision-induced dissociation. This ligation-based strategy for enhancing LC-MS/MS characterization of RNA creates opportunities to measure low abundance RNA samples and their modifications.

PurposeTo present the design and validation of a lowcost, microcontrollerbased gas delivery system that automates fixed inspired respiratory stimuli for MRI experiments. MethodsThe system uses three solenoid valves controlled by an Arduinobased circuit to switch between premixed medical gas cylinders according to predefined timing protocols. By using the MRI scanner external timing signal, gas delivery can be synchronised with image acquisition. Both a permanently installed configuration and a portable enclosure were constructed using commercially available components, with a total material cost of approximately {pound}650. The system was integrated with a singleuse breathing circuit and evaluated using hypercapnic and hyperoxic stimulus paradigms. Endtidal oxygen and carbon dioxide were measured using a respiratory gas analyser and physiological responses were assessed using BOLD MRI at 3 T. ResultsThe system delivered reliable, repeatable gas transitions during MRItriggered protocols. During hypercapnia (n{square}={square}15), the mean increase in endtidal carbon dioxide was 8.7{square}{+/-}{square}1.8{square}mmHg from a baseline of 32.2{square}{+/-}{square}3.1{square}mmHg, producing a mean grey matter BOLD signal increase of 3.2{+/-}1.7%. During hyperoxia (n{square}={square}15), the mean increase in endtidal oxygen was 292.3{square}{+/-}{square}59.0{square}mmHg from a baseline of 114.5{square}{+/-}{square}10.7{square}mmHg, with an associated BOLD signal change of 1.2{+/-}1.7%. Across both protocols respiratory and BOLD responses were consistent across participants. ConclusionThis microcontrollerbased system provides an inexpensive and reliable method for administering fixed inspired respiratory stimuli with automated MRI synchronisation. It offers an intermediate option between simple manual systems and higher cost commercial gas blenders, making it well suited for technical and methodological studies in cerebrovascular reactivity, hyperoxiaBOLD and related applications.