Analytical Biochemistry

High dietary fructose consumption is a major contributor to the development of obesity and related cardiometabolic diseases, highlighting the need for accurate assessment of fructose metabolism in humans. Stable isotope tracer approaches, such as 13C6-fructose, require highly sensitive and specific analytical methods to quantify both concentrations and isotopic enrichments. In this study, we developed and validated a robust gas chromatography-triple quadrupole mass spectrometry (GC-QQQ)-based method for the simultaneous measurement of unlabeled and 13C6-fructose in human plasma. The method employs oximation and per-acetate derivatization, and demonstrates high specificity and accuracy. Intra- and inter-assay precision were below 10%, with no detectable carry-over, and a lower limit of quantification (LLOQ) of 0.1 nmol/mL for concentration and 0.02 molar percent excess (MPE%) for enrichment and no interference from glucose. We further compared data acquisition using multiple reaction monitoring (MRM) and selected ion monitoring (SIM). MRM showed superior performance at the low concentrations and enrichment levels characteristic of clinical plasma samples, resulting in improved sensitivity and lower LLOQs compared to SIM. Overall, this validated method provides a sensitive and reliable approach for fructose tracer studies in humans. Its application will facilitate robust investigations into fructose metabolism, and its role in metabolic dysregulation and obesity-related disease.

Obtaining sufficient amounts of pure protein for downstream applications such as native mass spectrometry (nMS) is often challenging, especially when expression yields are low or proteins are unstable. In these cases, the commonly required buffer-exchange step is a major bottleneck, as it often leads to substantial protein loss and compromises biophysical characterization. These challenges are exacerbated in insect or eukaryotic expression systems, where protein yields are typically lower than in bacteria, making protein loss during purification particularly detrimental. Standard lysis and purification buffers contain non-volatile components such as Tris, phosphate, HEPES and sodium chloride, which form adducts during electrospray ionization (ESI) interfering with the signal and therefore must be re-moved prior to nMS. To address protein loss associated with this mandatory buffer-exchange, we evaluated an affinity-purification workflow, in which non-volatile salts are excluded throughout purification and proteins are directly eluted into nMS-compatible ammonium acetate-based buffers. This approach eliminates the need for a separate buffer exchange step and enables rapid nMS analysis immediately after affinity purification. We show that common eluents used in His- and Strep- based affinity purification, such as imidazole, biotin, and desthiobiotin, are well tolerated at relevant concentrations, allowing acquisition of high-quality spectra suitable for determining protein stoichiometry and for monitoring enzymatic or assembly processes. Together, this fast-track affinity workflow increases protein recovery, shortens sample preparation and complements online exchange protocols, which are less suited for monitoring processes. It hence expands the applicability of nMS to proteins and protein complexes that are difficult to obtain in sufficient quantity using conventional purification and buffer exchange strategies.

Precise quantification of myofilament protein phosphorylation is essential for understanding the regulation of cardiac contractility in health and disease. Although Phos-tag SDS-PAGE is widely used to resolve phosphorylated protein isoforms, its reproducibility and quantitative reliability are often limited by variability in the key experimental factors, including gel composition, electrophoretic conditions, protein loading, and sample preparation. Here, we present a standardized manganese (Mn2+)-Phos-tag SDS-PAGE workflow optimized for cardiac myofilament proteins, using myosin regulatory light chain 2 (MLC2) and cardiac troponin I (cTnI) as model targets. We systematically evaluated critical parameters - including Mn2+ and Phos-tag concentrations, acrylamide composition, electrophoretic regime, buffer chemistry, protein loading, and EDTA-mediated transfer - to define conditions that maximize phospho-species resolution while preserving quantitative fidelity. We further demonstrate that electrophoresis rate, sample loading, and extraction strategy significantly influence band morphology, signal intensity, and the apparent distribution of phospho-species. As a use case scenario, we compared Trichloroacetic acid (TCA) extracted mouse left ventricular homogenates with myofibrils prepared using a widely adopted Triton-X-100 tissue-demembranization protocol. Myofibril preparation was associated with profound MLC2 dephosphorylation at the earliest stages of preparation, whereas cTnI exhibited a marked reduction in higher-order, low-stoichiometry phosphoforms. Further evaluation of Myosin-binding protein C (MyBP-C) showed progressive loss of phosphorylation over the course of 24 hours. We submit that TCA-extracted heart standards in combination with Phos-tag gels can provide valuable quality control for the phosphorylation status of myofibril preparations, and that inclusion of a high-affinity PP2A and PP1 phosphatase inhibitor like okadaic acid may benefit future myofibril mechanics studies.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Background: Acute phase response is an early immunometabolic response to brain injuries, primarily coordinated by the liver via the activation of acute phase proteins. These immune responses can be both beneficial, promoting tissue repair, and detrimental, exacerbating neurological deficits, if not properly controlled. Despite the central role of the liver in immunometabolism, how hepatic metabolism dynamically adapts to traumatic brain injury remains under explored, primarily due to limited liver-specific modalities that can assess metabolic pathways in vivo. 13C MRI utilizing hyperpolarized 13C-pyruvate can assess key regulatory enzyme activities in hepatic metabolism. Methods: Rats with controlled cortical impact were studied in vivo using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate under fed and fasted conditions 3-4 days after injury. Hyperpolarized 13C products, including [13C]bicarbonate from [1-13C]pyruvate and [5-13C]glutamate, [1-13C]acetyl-L-carnitine, and [2-13C]phosphoenolpyruvate from [2-13C]pyruvate, were evaluated to assess mitochondrial and gluconeogenic metabolism. In parallel, liver tissues were collected following [U-13C3]pyruvate injection for NMR isotopomer analysis of phosphoenolpyruvate, glucose, and glutamate. Results: While no metabolic differences were detected under fed condition, [13C]bicarbonate and [2-13C]phosphoenolpyruvate increased after brain injury under fasted condition, indicating an upregulation of the hepatic gluconeogenic pathway after injury. 13C NMR of liver tissue extracts from injured rats showed an elevated [2,3-13C2]glutamate-to-[4,5-13C2]glutamate ratio and increased 13C-labeling in phosphoenolpyruvate than controls, confirming enhanced hepatic gluconeogenic pathway. Conclusion: This study demonstrates that hepatic acute phase response to brain injuries can be monitored in vivo by hyperpolarized pyruvate, which may be further utilized for longitudinal immunometabolic evaluation of the liver during pathogenesis and therapeutic interventions.

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.

Brook trout (Salvelinus fontinalis) exhibit strain-level variation in growth performance, environmental tolerance, and survival, yet the biochemical mechanisms underlying these differences remain poorly understood. We developed and applied a ratiometric biochemical framework integrating the pentose-phosphate pathway (PPP) and glutathione metabolism to characterize strain-specific hepatic metabolic organization in brook trout. Five strains reared under standardized conditions differed significantly in hepatic soluble protein density, glutathione pool size, total NADP(H) concentration, and activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and transketolase (TKT). These differences were not uniformly coordinated across pathways, demonstrating that metabolic phenotype cannot be inferred from individual biomarkers alone. Derived ratios describing oxidative-to-non-oxidative PPP capacity (G6PDH/TKT) and glutathione buffering relative to recycling capacity ((GSH+GSSG)/GR) resolved distinct patterns of metabolic allocation among strains. Despite shared ancestry, the Temiscamie (TEM) strain and its domestic x TEM hybrid (TXD) exhibited markedly divergent metabolic phenotypes, demonstrating that closely related strains can differ substantially in hepatic metabolic organization. Together, these findings identify relative allocation among interconnected metabolic pathways as an axis of physiologic diversity and establish a ratiometric approach for comparing metabolic organization across populations and species. Graphical abstractHepatic metabolic phenotypes of brook trout strains were characterized by integrating pentose phosphate pathway enzyme capacities, glutathione metabolism, NADP(H) availability, and soluble protein into a ratiometric framework. Ratios distinguish investment in oxidative versus non-oxidative PPP capacity (G6PDH/TKT), antioxidant buffering versus glutathione recycling capacity (total glutathione/GR), and hepatic protein density (soluble protein/liver mass), revealing distinct metabolic organization among strains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/743818v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1694676org.highwire.dtl.DTLVardef@90f2d4org.highwire.dtl.DTLVardef@365327org.highwire.dtl.DTLVardef@8d56ca_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA ratiometric framework was developed to characterize hepatic metabolic organization in brook trout C_LIO_LIGlutathione buffering and recycling capacity distinguish alternative redox phenotypes C_LIO_LIInvestment in oxidative and non-oxidative PPP capacity varies independently among strains C_LIO_LIG6PDH/TKT and total glutathione (GSH+GSSG)/GR reveal distinct metabolic phenotypes C_LIO_LIRatiometric indices provide a framework for interpreting redox metabolism and carbon allocation C_LI

BACKGROUND: Circulating lipoprotein measures such as low-density lipoprotein (LDL) cholesterol and apolipoprotein B are causal biomarkers of cardiovascular risk. These can be quantified quickly and accurately by nuclear magnetic resonance (NMR) spectroscopy, but clinical translation has been slow. We investigated easy-to-operate and affordable benchtop NMR technology as a new means to quantify lipoprotein biomarkers in point-of-care settings. METHODS: Serum samples from 336 individuals were analysed with a benchtop NMR spectrometer operating at 80 MHz and a high-field NMR spectrometer operating at 600 MHz. Glucose, apolipoprotein A-I, apolipoprotein B, total triglycerides, total cholesterol, LDL cholesterol and high-density cholesterol were determined by standard biochemistry. Corresponding NMR-based measures were quantified by linear regression. The 80 MHz dataset included experiments with different scan settings to optimize measurement time (1,987 spectra in total). RESULTS: We identified 32 scans (2 min 8 s) as the minimum runtime for lipoprotein quantification. Total triglycerides and glucose were quantified with the highest accuracy (CV [≤]5.2%, R2 [≥]95%), while LDL cholesterol was more challenging (CV = 10.1%, R2 = 72%) and apolipoprotein B in between (CV = 7.4%, R2 = 74%). Epidemiological correlations between biochemistry assays and sex, obesity, glycemia and blood pressure were reproduced by the corresponding benchtop assays (P [≥]0.11 for difference). CONCLUSIONS: We developed a new lipoprotein quantification method and demonstrated its feasibility for standard lipoprotein analytics. The portability and cost-effectiveness of benchtop NMR make it an appealing choice for research and clinical settings where rapid and robust results on site are an advantage.

We present a simple and efficient way to add cis dialdehydes, phosphoryl groups, or cis diols to the 3 prime end of oligonucleotides using periodate oxidized nucleotides (oNTPs) and terminal deoxynucleotidyl transferase (TdT). The 3 prime end cis dialdehyde-modified oligos are generated by incubating TdT with an oligo for several minutes followed by addition of a oNTP and incubated at 30 degrees C for 30 minutes to an hour. After allowing the addition of the cis dialdehydes, heating the reaction mixture at 90 to 95 degrees C for 10 minutes yields oligonucleotides with 3 prime phosphoryl groups. The 3 prime cis diol modified oligos are synthesized by starting with 3 prime cis diol nucleotides (HO-NTPs). The cis dialdehyde modified oligonucleotides and cis diols may then be used for a variety of investigations such as studying the interaction of proteins with the 3 prime end of DNA, and possibly RNA. As an example, we demonstrate the efficacy of using an oligonucleotide modified with oGMP at the 3 prime end as an affinity label for TdT and identified a peptide fragment that has been shown to contain two of three aspartate residues found to be in the TdT active site.

Background. Roughly, half of new type 1 diabetes (T1D) diagnoses occur in individuals under 18 years old and represent a more aggressive destruction of beta cell mass (BCM). [11C]-(+)-PHNO positron emission tomography (PET) imaging is used to assess BCM, but current pancreas PET imaging protocols are limited to adults. Previously published full count data from six healthy controls and five T1Ds (6M/5F; 22 to 53 years old) were used for retrospective analysis. Dynamic [11C]-(+)-PHNO PET/CT scans were acquired and reconstructed using full-count list-mode data. For the current comparison to full count data, 50%, 25% and 10% down-sampled count data were re-reconstructed. Pancreas and spleen (reference region) time-activity-curves (TACs) were assessed, and volume of distribution (VT, mL/cm3) was estimated using the reversible 1-tissue compartment model (1TC) with tmax of 30 min for all count levels. Pancreas and Spleen VT estimates (1TC; tmax= 30 min) were used to calculate non-displaceable binding potential (BPND) and were then correlated to semi-quantitative methods of standardized uptake value ratio (SUVR-1) (20-30 min; ref: spleen) to examine simplified methods using simulated low dose protocols. Finally, we performed dosimetry in adult, adolescent and pediatric phantoms to assess radiation dose for simulated low-dose protocols. Results. Qualitatively, increasing noise can be visualized at successive reduced-count levels images, compared to full-count images. Despite progressively increasing noise in reduced-count images, TACs at each reduced-count level remained similar to full-count TACs in both HC and individuals with T1D. Quantitatively, 1TC VT estimates were similar for all reduced count levels and range of tmax values, compared to full-count (all R2[≥]0.99). Pancreas SUVR-1 (20-30 min) and pancreas BPND (tmax = 30; ref: spleen) were highly correlated for all count levels (all R2[≥]0.80). All age groups were under both the yearly occupational and research scan radiation dose limits when examining mean effective dose equivalent with reduced (1/10th) injected dose protocols. Conclusion. Low-count reconstructed data and simplified reference region approaches provide accurate quantification compared to full-count reconstructions. These results provide evidence that it is possible to perform accurate quantification using simulated low dose protocols to quantify BCM for use in individuals with T1D under 18 years old.

Recombinase polymerase amplification (RPA) enables rapid nucleic acid testing in low-resource environments, but poorly characterized byproducts can compromise assay specificity and cause false-positive results. Here, we amplified the thirteen original CODIS core loci and Amelogenin to characterize recurrent RPA artefacts and establish conditions that reduce their formation. First, RPA products were analyzed for two reference samples by Oxford Nanopore Technologies sequencing. This revealed two distinct classes of multimeric products: primer multimers and amplicon multimers, consisting of repeated primer or amplicon sequences, respectively. Individual artefacts contained up to 281 primer copies or 22 amplicon copies, demonstrating the extensive range of these products. Next, we performed an optimization study to evaluate the effects of reaction temperature and reagent concentrations at two representative loci, D3S1358 and D5S818. Among the conditions tested, temperature had the most pronounced effect. Reducing the temperature from 42{degrees}C to 34{degrees}C increased the relative target amplicon fraction from 15% to 83% for D3S1358 and from 84% to 98% for D5S818, while maintaining or increasing absolute target concentration. Lower primer concentrations and higher T4 UvsX concentrations also reduced multimer formation, although lower primer concentrations reduced target yield and caused allelic dropout. Finally, amplification at 34{degrees}C was evaluated across all fourteen loci by sequencing. Relative to 42{degrees}C, the target read fraction increased by more than 5 percentage points for 7/14 loci in one reference sample and 9/14 loci in the other, with the largest improvements at multimer-prone loci. These findings identify multimers as an important class of RPA artefacts and establish reaction temperature and T4 UvsX concentration as promising conditions to improve RPA specificity.

Developments in Nanopore sequencing have enabled telomere to telomere genomic assembly as a routine technique in genomic research. Nanopore DNA sequencing for genomic assembly is typically performed on native DNA molecules, making it particularly sensitive to the quality of input DNA, with contaminating molecules limiting data yields and reducing read quality. As pangenome analysis gains interest, particularly in non-model plant species which are often rich in inhibitory secondary metabolites, the development of methods which can improve the quality and throughput of nanopore sequencing is essential. Here we describe a method for isolation of total DNA from the leaf tissues of diverse Viridiplantae species. The initial lysis buffer consists of a modified CTAB buffer, incorporating dimethyl sulfoxide for the reduction of viscosity, which can be problematic in many plant DNA preparations. An organic extraction with 2-butoxyethanol is utilized to further extract phenolic compounds which may be sufficiently hydrophilic to evade chloroform extraction, while reducing aqueous phase volume. Further cleanup via cesium chloride (CsCl) ultracentrifugation is performed to minimize the carryover of residual contaminating macromolecules. Samples prepared using this method are of consistent high quality, even when extracted from challenging late season leaf tissue or secondary metabolite rich species. Sequencing results from samples prepared by this method outperform those obtained from typical modified CTAB DNA isolation techniques in both quantity and quality. We tested sequencing performance from Vitis DNA isolated using a modified CTAB method and Vitis DNA isolated using the CsCl ultracentrifugation-based method described here. DNA isolated via the method described here produced 83% more >Q10 sequence data (52.61 Gb vs. 28.8 Gb), resulted in a 60% greater read N50 despite more handling steps (32.78kb vs. 20.45kb), and resulted in a higher modal read quality (Q27 vs. Q24). The consistency of this method across diverse plant taxa suggests its use as a general method for DNA isolation prior to Nanopore sequencing and genomic assembly for diverse plant taxa.

Objective To evaluate the effect of vendor-integrated AI-assisted abdominal ultrasound software on operational efficiency and sonographer workload compared with manual scanning. Methods In this prospective randomised crossover study (January to February 2026), 32 healthy adults each underwent two upper abdominal examinations, one manual and one using vendor-integrated AI software (AI Abdomen Release 3.5; ACUSON Sequoia), in randomised order by two experienced sonographers, each participant scanned once by each sonographer. Scan time, hand-console interaction (keystrokes, hand travel, hover, jerk) from a custom depth-camera hand-tracking system, and operator modifications to AI outputs were recorded. Workload was assessed after each scan with the weighted NASA Task Load Index (NASA-TLX). Analysis used linear mixed-effects models. Results AI-assisted scanning reduced scan time (52.4 s, approximately 9%; 95% CI 23.7 to 81.2; P = 0.001), keystrokes (55, approximately 28%; P < 0.001) and hand travel (4.57 m, approximately 39%; P < 0.001), although the time saving was concentrated in one sonographer. Weighted NASA-TLX did not differ between conditions (-3.9 points; 95% CI - 9.3 to 1.5; P = 0.17), but subscale analyses showed reductions in mental demand (- 6.3; P = 0.03) and effort (- 7.0; P = 0.04), with no compensating increases. Sonographers modified 48 of 184 AI-generated values. Conclusion AI assistance improved operational efficiency and reduced self-reported mental demand and effort, with no compensating increase on other subscales. Gains arose under a controlled, abbreviated protocol in healthy volunteers and varied between operators, and are better read as a reshaping of operator work than its removal.

Mass spectrometry (MS)-based nucleic acid analysis provides direct chemical evidence for oligonucleotide sequence, composition, and modifications. However, oligonucleotide LC-MS analysis commonly relies on ion-pairing reversed-phase liquid chromatography (IP-RPLC). Although IP-RPLC provides strong retention and high-resolution separation of highly charged nucleic acids, ion-pairing reagents can contaminate LC-MS systems, suppress electrospray ionization, require extensive system cleaning, and limit the use of high-end MS platforms that are primarily dedicated to proteomics or metabolomics. Here, we developed and evaluated an ion-pair-free capillary hydrophilic interaction liquid chromatography mass spectrometry (capillary HILIC-MS) workflow for RNA modification mapping. To enable robust analysis of biologically relevant samples, we optimized sample preparation, high-organic loading conditions, chromatographic parameters, and MS source settings to overcome key challenges associated with capillary HILIC, including limited sample volume, solvent compatibility, and solvent breakthrough during injection. The optimized capillary HILIC-MS method provided effective separation of oligonucleotides below 30 nt and enabled sensitive detection of RNA modifications in the populations of tRNAs and rRNAs in biological samples. Importantly, the ion-pair-free workflow also allowed switching between nucleic acid analysis and proteomics on the same LC-MS platform without the need for extensive system decontamination. Together, this workflow provides a sensitive, robust, and MS-compatible approach for nucleic acid analysis, expanding the utility of high-end LC-MS systems for both therapeutic oligonucleotide characterization and biological RNA modification profiling.

BackgroundSingle-voxel magnetic resonance spectroscopy (MRS) is a non-invasive method for measuring clinically and cognitively relevant metabolites. Reliable measurements require precise voxel placement across sessions and participants. We developed a scanner-console-based approach to improve voxel placement precision. MethodsIn a crossover design (n=7; six sessions each), we compared test-retest reliability of three voxel placement methods in a reference benchmark (left parietal cortex) and a technically challenging region (left ventromedial prefrontal cortex). Methods included (1) conventional anatomy-based placement, (2) mask-guided real-time positioning (MGRP), and (3) semiautomated session-locked voxel repositioning (SSVR). Resting-state MRS data were acquired using PRESS and MEGA-PRESS. Within-subject reliability of voxel placement and metabolite concentrations, namely, total N-acetylaspartate (tNAA), total Creatine (tCr), GABA (gamma-aminobutyric acid), and Glx (glutamate + glutamine) are reported using the coefficient of variation (CV), the intraclass correlation coefficient (ICC), minimal detectable change (MDC), and the spatial overlap. ResultsSSVR markedly improved voxel placement reliability, increasing spatial overlap (up to 88%) and achieving near-perfect geometric reproducibility (ICC = 0.99) compared to conventional anatomy-based placement and MGRP. SSVR improved tissue composition consistency and reduced metabolite variability in the technically challenging region (variability reduction of [~]70% tCr, [~]59% tNAA, and [~]51% Glx) while further refining already stable measurements in the benchmark region (tNAA from [~]15% to [~]10%). ConclusionBoth MGRP and SSVR improved voxel placement and metabolite measurement reproducibility compared with conventional anatomy-based placement. SSVR further enhanced within-subject reproducibility across repeated sessions, particularly in the technically challenging region, providing a robust approach for longitudinal single-voxel MRS studies.

Acetyl-CoA and other acyl-CoA thioesters are critical intermediates in the metabolic reactions that cells rely on to produce energy and carry out biosynthesis. Therefore, the analysis of acyl-CoA provides valuable information about the metabolic activity of cells, especially when combined with stable isotope tracing. Acyl-CoA species are routinely monitored by reversed-phase liquid chromatography coupled to tandem mass spectrometry (RPLC-MS/MS). However, drastic differences in the hydrophobicity of short-chain versus long-chain acyl-CoA species have been challenging to accommodate with a single set of RPLC conditions. Here, we describe a convenient method based on hydrophilic interaction liquid chromatography (HILIC-MS/MS) for the concurrent detection of both short-chain and long-chain acyl-CoA and their corresponding acyl-carnitine species. Using this strategy, we tracked the metabolism of isotope-labeled fatty acids in multiple cell lines, which revealed differences in their propensities for fatty acid oxidation and the extent to which isotope incorporation into acyl-CoA mirrored that of acyl-carnitine. We also applied the HILIC-MS/MS workflow to the analysis of NADH and ATP, making it a useful technique for gauging cellular bioenergetics as reflected by the acetyl-CoA/CoA, NADH/NAD+, and ATP/ADP ratios. Altogether, this HILIC-MS/MS platform enables a streamlined analysis of acyl-CoA species and other key intermediates in cell metabolism.

Background Even a highly accurate diagnostic test can yield more false-positive than true-positive findings in low-prevalence settings, which is known as the false positive paradox. Radiologists' unawareness of this paradox may foster automation bias, the tendency to excessively rely on AI outputs. Methods In this prospective, multinational, randomized controlled reader study (DRKS00038740), 34 readers from 10 countries (16 residents, 8 general radiologists or fellows, and 10 neuroradiologists) were randomly assigned to a control group (n = 17) or intervention group (n = 17), stratified by experience level. The intervention group reviewed a short, 3-minute educational video explaining the false positive paradox prior to the reading session. Both groups evaluated 20 TOF-MRA studies with AI-flagged findings (10% true-positive, 90% false-positive). Primary outcomes were acceptance rate of false-positive AI findings and follow-up intensity. These were evaluated using mixed models with crossed random effects for reader and case. Results At baseline, readers vastly overestimated the positive predictive value of AI tools for intracranial aneurysm detection (mean estimate, 62.9%; simulation-based estimate, 15.4% [95% interval, 8.1-28.0%]). The intervention reduced the odds of accepting AI false positives (OR 0.50 [upper 95% confidence bound, 0.95], one-sided p = 0.017), with acceptance probabilities of 12.7% (95% CI, 6.0-25.0%) in the intervention group compared to 22.5% (95% CI, 11.6-39.2%) in the control group. The intervention group exhibited a downward shift in follow-up intensity for false positives (OR 0.47 [upper 95% confidence bound, 0.81]; one-sided p = 0.014), recommending follow-up in 39.2% (120/306) of cases, compared to 54.9% (168/306) in the control group. Conclusion A brief education on the false positive paradox improved trust calibration in AI-assisted intracranial aneurysm detection. Our findings highlight the potential of reader-side cognitive debiasing strategies to improve trust calibration and support safer use of AI in radiology.

Type 2 diabetes (T2D) is a prevalent metabolic disorder affecting millions worldwide, characterized by insulin resistance and impaired glucose homeostasis. While mammalian models are widely used, Drosophila melanogaster provides a powerful alternative due to its conserved insulin signaling pathways, genetic tractability, and suitability for high throughput studies. In addition to glucose dysregulation, lipid metabolism plays a crucial role in T2D pathophysiology, as alterations in lipid composition contribute to insulin resistance and metabolic dysfunction. Lipidomic studies have emerged as an essential approach to identify metabolic signatures and potential biomarkers for disease progression and therapeutic targeting. In this study, T2D like model was established by inducing insulin resistance through knockdown of the insulin receptor in brain insulin-producing cells using the dilp2-Gal4>UAS-InRRNAi system. This genetic manipulation resulted in significant metabolic dysregulation, including elevated glucose, trehalose, and triacylglyceride levels, along with increased oxidative stress indicators. Additionally, mRNA expression analysis of key insulin signaling components, including insulin receptor substrate 1, dilp2, dilp3, dilp5, and phosphorylated Akt, further validated the model. To further investigate metabolic alterations, Lipid profiling was performed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) in non targeted LC-MS-based metabolomics approach to identify lipid biomarkers associated with T2D. Multivariate statistical analyses, including PCA and PLS-DA, revealed distinct lipid signatures between wild-type and T2D flies. Notably, specific phosphatidylglycerol species PG 34:0, PG 34:4, PA 38:3, PIP 38:1, PIP2 38:6, and LPS 24:0 demonstrated an area under the curve (AUC) of 1, indicating their strong reliability as lipid biomarkers for T2D diagnosis.

Molecular specificity remains a major challenge in mass spectrometry imaging (MSI), particularly when low-abundance species coexist with structurally related isomers that cannot be distinguished by accurate mass and exhibit similar fragmentation behavior. Bis(monoacylglycero)phosphates (BMPs), lysosomal lipids increasingly implicated in lipid homeostasis and disease, represent a particularly demanding example because they are structural isomers of phosphatidylglycerols (PGs) and display highly similar negative-ion fragmentation. Here, we developed an ion mobility-guided targeted MALDI-MS/MS imaging workflow for direct on-tissue discrimination of endogenous BMP/PG isomeric pairs. Orthogonal HILIC-DDA-PASEF analysis provided accurate-mass, retention-time, fragmentation, and ion-mobility information used to define mobility-constrained precursor coordinates for scheduled MALDI-iPRM-PASEF acquisition. Ion-mobility measurements showed high agreement across ESI-TIMS, MALDI-TIMS, and tissue-based MALDI-TIMS-MSI, while optimization of laser sampling minimized ion-load-dependent mobility shifts. Narrow mobility windows reduced reciprocal PG/BMP cross-talk to below 4% while preserving selective detection under strongly unbalanced abundance conditions. The workflow enabled distinct precursor- and product-ion imaging of endogenous PG 34:1 and BMP 34:1 in sagittal mouse brain, supporting their acyl-chain-level assignment as PG 16:0_18:1 and BMP 16:0_18:1. Application to a CLN3-knockout mouse model revealed BMP-specific reductions across brain, kidney, and lung that were not mirrored by the corresponding PG isomers, providing an orthogonal biological validation of the analytical discrimination. Mobility-constrained targeted MS/MS additionally resolved type-II isotopic interference that remained ambiguous at the MS1 level. Overall, this work provides a strategy for reciprocal spatial discrimination and structural confirmation of endogenous BMP and PG isomers directly in tissue and highlights the value of combining ion mobility with targeted product-ion imaging to increase molecular specificity in spatial lipidomics.

External quality assessment (EQA) of multianalyte assays is commonly interpreted analyte by analyte, although many panels contain known relations among measured features that may reveal joint quality patterns. We propose PathEQA, a feature-graph-guided random forest framework in which a user-supplied graph can represent biochemical pathways, molecular interactions, shared measurement processes, or other domain relations. The same graph is allowed to influence feature representation, node-level candidate generation, and split selection, with an optional local grouped decision. We evaluated the framework in graph-aligned and graph-misspecified simulations and used a six-analyte catecholamine-related liquid chromatography-tandem mass spectrometry EQA data set as an illustrative case study (929 records from 58 laboratories and 117 complete multianalyte panels). In graph-aligned simulations, the grouped variant reduced test root mean squared error by 7.4-9.4% relative to ordinary random forest across training sizes of 60-240, whereas graph misspecification could worsen prediction. In the catecholamine case study, full PathEQA was comparable with ordinary random forest in laboratory-grouped cross-validation (RMSE 0.570 versus 0.569) and modestly better in the final-round temporal holdout (0.307 versus 0.318); a simpler static network-sampling baseline performed best. Dopamine-norepinephrine was the strongest pair, whereas dopamine-norepinephrine-epinephrine best estimated multianalyte failure burden. These results support a general conclusion: feature-graph guidance can improve small-sample multivariate quality assessment when the supplied structure is outcome-relevant, but graph relevance must be tested rather than assumed. Catecholamines serve here as a worked example rather than a restriction of the framework.