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.

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.

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

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.

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.

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.

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.

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein mass range (17-800 kDa). We analysed holo-myoglobin (17 kDa) over a range of concentrations (10 M-10 nM) and capillary voltages to determine limits of detection and define a gentle operating regime. We additionally observe reduced Na+ adduction and preservation of the Zn2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin ([~]44 kDa), malate dehydrogenase ([~]70 kDa), glutamate dehydrogenase ([~]350 kDa), {beta}-galactosidase ([~]465 kDa), and GroEL ([~]800 kDa), were readily detected using nanopipette emitters. Compared with conventional 1-2 m internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of clarified bacterial lysate expressing -synuclein yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

Metabolomics offers a sophisticated analytical framework for characterising the molecular phenotype of biological organisms and complex living systems at a high resolution. As the functional endpoint of the omics cascade, the metabolome serves as a close reflection of cellular activity. It integrates genetic, transcriptomic and proteomic variations with external environmental influences. However, the inherent complexity of metabolomic datasets, characterised by high-dimensional chemical diversity, wide dynamic ranges, and significant matrix effects, necessitates a rigorous suite of chemometric and bioinformatic workflows. For researchers uninitiated in computational biology, the multi-stage requirement for raw data pre-processing, signal deconvolution, and multivariate statistical modelling (such as PCA or PLS-DA) presents a substantial barrier to entry. Navigating these convoluted data architectures remains a primary challenge in deriving biological meaning from the global metabolic profile. Here, we present a workflow to use Python Dash Apps to create a user-friendly interface for simplifying data processing and statistical calculations. Users can select their desired samples to initiate calculations for various statistical tests, generating interactive and publication-quality figures to explore their results. These apps were deployed on an Apache server via cPanel, allowing individuals to share their findings with collaborators and for research facilities to share metabolomics results with their users.

Background: Traditional bone scintigraphy for detecting malignant bone metastases is limited by suboptimal accuracy and radiation exposure. Whole-body magnetic resonance imaging (WB-MRI), while an alternative, requires lengthy scan times and high patient compliance. Purpose: To develop a novel, rapid whole body bone screening (WB-RBS) MRI protocol and evaluate its diagnostic performance for bone metastasis detection. Materials and Methods: Patients with pathologically confirmed malignancies and healthy controls were prospectively enrolled. All participants underwent WB-RBS (acquisition time: about 10 min); patients additionally underwent WB-MRI (about 70 min). Three radiologists, blinded to clinical data, independently evaluated the images for bone metastases. A consensus expert diagnosis served as the reference standard to calculate the diagnostic performance of WB-RBS. Specificity was further assessed in the healthy control group. Results: Seventy patients and 19 healthy controls were included. WB-RBS demonstrated excellent inter-reader agreement at the patient level. Compared with the reference standard, WB-RBS achieved an accuracy of 77.1%-91.4% at the patient level and a slightly lower accuracy (70.6%-82.5%) at the lesion level. At diagnostic confidence thresholds 1-3, the correlations between WB-RBS ratings and the reference standard were statistically significant for both patient- and lesion-level analyses. Conclusion: WB-RBS showed favorable inter-reader agreement and high accuracy for bone metastasis screening at the patient level, while substantially reducing scan time and cost. Its rapid, radiation-free nature and high accessibility offer distinct clinical advantages, supporting its potential as an alternative screening tool to conventional bone scintigraphy.

PurposeTo address the limitations of single-distance, 1D performance metrics in RF coil design. This work introduces a multi-objective, volume-of-interest (VOI) based analysis to systematically characterize the trade-offs between power efficiency, pSAR efficiency, and homogeneity as a function of dipole length (l) and distance-to-load (d) for multiple dipole geometries and target anatomies. MethodsElectromagnetic simulations of straight and end-meandered dipole antennas were performed with varying lengths (100-500 mm) and distance-to-load (1-81 mm) over three anatomical targets (prostate, kidney, heart). Homogeneity, power efficiency, pSAR efficiency, and load sensitivity performance metrics were calculated within each anatomical VOI. Inter-element coupling at variable d was assessed in a 3-element array, and a subset of single-element simulations was experimentally validated using B1+ mapping. ResultsA fundamental trade-off was found between power efficiency and pSAR efficiency. Optimal power efficiency was achieved with shorter dipoles (150 mm < l < 300 mm) closer to the sample (d < 30 mm), while optimal pSAR efficiency and homogeneity were achieved with longer dipoles at further from the sample (d > 60 mm). Inter-element coupling increased with distance-to-load but could be managed by increasing element spacing. Experimental measurements were in good agreement with simulation trends. ConclusionIncreasing distance-to-load to 40-60 mm, compared with commonly used distances of 20-30 mm, offers a practical strategy for improving pSAR efficiency and homogeneity with a minimal decrease in power efficiency. This work provides a quantitative analysis that enables RF coil designers to make informed, data-driven decisions when developing next-generation body arrays and suggests that unshielded end-meandered dipoles could be an optimal transmit element geometry.

Nanopore read-out, that is the current signals measured across nanometer-sized openings in dielectric membranes or through natural protein channels, enables the detection, identification and sequencing of individual molecules. The detection can take place by analyzing the events of single biomolecules interacting with the pore. The accuracy in the detection of these single events is key for identification of physicochemical properties of analyte molecules. To this end, we further develop a very simple, fast, almost parameter-free, and adaptable cluster-based event detection (CBED) algorithm that clusters the nanopore signals prior to detecting nanopore events. The algorithm is validated against two other event detection schemes with respect to simplicity and efficiency. For this, nanopore data from four different experiments stemming from different laboratories that vary in the nanopore type, size, and analyte are considered. The comparison is made on the basis of the number of events detected, their quality, and the most important features extracted from nanopore events. Our results underline the higher efficiency and less noise of the CBED detected events for biological nanopore data and the need for an on-the-fly adaptivity of the baseline current for a class of solid-state nanopore data.

Assessment of pathologic complete response (pCR) following neoadjuvant chemotherapy (NAC) remains an unmet clinical need in breast cancer. Fibroblast activation protein inhibitor (FAPI) PET targets the tumor microenvironment and may therefore enhance response evaluation after NAC. This study aimed to compare the performance of [68Ga]Ga-FAPI-04 PET, [18F]FDG PET, and contrast-enhanced MRI for predicting pathologic response after NAC in breast cancer, with separate analyses for primary breast lesions and axillary lymph nodes. MethodsIn this prospective single-center diagnostic accuracy study, women with biopsy-confirmed stage II-III breast cancer underwent baseline and post-therapy [68Ga]Ga-FAPI-04 PET/MRI, [18F]FDG PET/CT, and contrast-enhanced MRI before surgery. Quantitative PET parameters were evaluated for primary tumors and axillary lymph nodes. pCR was defined as ypT0/isN0. Significant variables identified in univariable analyses were further explored using least absolute shrinkage and selection operator (LASSO) analysis, and receiver-operating-characteristic (ROC) analysis was performed to assess diagnostic performance. Fibroblast activation protein expression was also assessed by immunohistochemistry in paired pre- and post-therapy tumor specimens from a subset of patients. ResultsTwenty-four patients completed the study protocol, yielding 25 primary lesions and 44 metastatic lymph nodes across 27 axillary compartments. Overall patient-level pCR was achieved in 13 of 24 patients (54.17%). The lesion-level pCR rate was 60.00% (15/25) for primary breast lesions, and the node-level pCR rate was 72.73% (32/44) for axillary lymph nodes. For primary tumor response, post-therapy [68Ga]Ga-FAPI-04 SUVmax showed the highest diagnostic performance (AUC, 0.84; sensitivity, 80.00%; specificity, 80.00%; accuracy, 80.00%), whereas the optimal [18F]FDG parameter was {Delta} TBR% (AUC, 0.747). For nodal response, post-therapy [68Ga]Ga-FAPI-04 SULmean showed the highest diagnostic performance (AUC, 0.89; sensitivity, 91.67%; specificity, 81.25%; accuracy, 84.09%) and was significantly different from the best [18F]FDG parameter ({Delta} SULmax%, AUC, 0.669) on DeLong testing (P < 0.05). MRI achieved AUCs of 0.733 for primary lesions and 0.770 for lymph nodes. Stromal FAP expression positively correlated with [68Ga]Ga-FAPI-04 SUVmax and was markedly reduced in lesions achieving pCR. ConclusionPost-therapy [68Ga]Ga-FAPI-04 PET may serve as a promising adjunctive imaging biomarker for predicting pathologic response after NAC in breast cancer, particularly for axillary nodal assessment. These findings suggest that FAPI PET may provide clinically relevant information for preoperative evaluation of residual disease burden, potentially contributing to more individualized surgical planning and treatment decision-making.

Background: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a genetic disease characterized by spasticity and ataxia which reflects involvement of the corticospinal tracts (CST) and cerebellum. The primary involvement of the middle cerebellar peduncles (MCP) and transverse pontine fibers (TPF) at the crossing with the CST, and their role in the pathophysiology of the disease, is currently debated. Objectives: Advanced MRI techniques capable of isolating sub-voxel microstructural parameters can test the hypothesis that the MCP and TPF are abnormally large, compressing the CST at their crossing, and potentially impairing CST development. Methods: Tract macro- and micro-structural properties, including axon and tract caliber, axon density and geometry, and myelin content were estimated from diffusion-relaxometry and magnetization transfer imaging. These features were analyzed along segments of the CST, MCP, and TPF of 9 patients and 9 age-matched controls. Results: While the CST showed significant decreases in tract size, axon caliber, and myelination throughout its length compared to controls (p<0.01), the MCP and TPF were relatively unaffected. In our group, neither the MCP nor the pons were enlarged. The proximal MCP showed an increase in axon caliber. Conclusions: The increase in fractional anisotropy and axon density towards the center of the TPF could be driven by geometric confounds related to differences in the relative sizes of the CST and TPF compared to controls. This highlights the importance of investigating tract-specific microstructural profiles, particularly in regions of geometric complexity. The findings confirm the involvement of the CST, with a relatively limited involvement of the MCP and TPF.

Many proteomics protocols rely on enzymatic digestion of complex protein mixtures to generate peptides with predictable cleavage patterns for the mass spectrometry analysis. One of the most utilized enzymes, trypsin, is classically defined as a serine endopeptidase with high specificity for cleaving peptide bonds on the C-terminal side of internal lysine and arginine residues. Accordingly, trypsin is not expected to remove the N-terminal arginine, which may arise through posttranslational modification such as arginylation or by proteolysis exposing internal residues as the new N-termini. N-terminal arginine plays important biological roles, including functioning as an N-degron and modulating protein interactions/signaling through its positive charge. Curiously, prior mass spectrometry-based studies utilizing trypsin to identify proteins bearing N-terminal arginine have frequently reported low and inconsistent yields, suggesting potential systematic bias in current proteomic approaches. Here, we explored whether trypsin would affect the integrity of the N-terminal arginine. By using antibodies specifically recognizing N-terminal arginine of different peptides, and by using mass spectrometry peptide analysis, we show that trypsin can remove N-terminal arginine residues in an exopeptidase-like manner. This effect occurs across a range of digestion conditions consistent with standard proteomic workflows, on peptides or whole proteins, and depends on trypsin concentration, incubation time, and catalytic activity. In addition, we show that the alternative arginine-cleavage enzyme Arg-C can also affect N-terminal arginine in a sequence-dependent context. In contrast, Lys-C and LysargiNase do not exhibit such effects, providing suitable alternative digestion strategies. Together, these findings reveal an unappreciated enzymatic behavior of arginine-cleaving proteases and suggest that their widespread use may systematically compromise the detection of N-terminal arginine in proteomic studies.

Objective:This study aims to compare the advantages and disadvantages of DLIR and adaptive statistical iterative reconstruction-V (ASIR-V) in thin-slice (2.5 mm) CT images of hepatic lesions characterized by high and low contrast. Additionally, the study seeks to determine the optimal DLIR strength for the evaluation of liver lesions. Methods:A retrospective analysis was performed on 90 patients who underwent abdominal contrast-enhanced CT scans. Group A comprised 48 patients with low-contrast lesions, while Group B included 42 patients with high-contrast lesions. The acquired images were reconstructed using post-processing DLIR at low (DLIR-L), medium (DLIR-M), and high (DLIR-H) strengths, all with a slice thickness of 2.5 mm (subgroups A1-A3, B1-B3). Furthermore, images were reconstructed with ASIR-V at 50% strength at slice thicknesses of 2.5 mm and 5 mm (subgroups A4/B4 and A5/B5, respectively). CT values and standard deviations (SD) of the liver and lesions were measured, and the corresponding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. The edge rise slope (ERS) was determined using ImageJ software by measuring CT values along a line from the liver parenchyma to the lesion. Objective metrics were compared using one-way ANOVA, with independent samples t-tests applied for inter-group differences. Subjective scoring, which encompassed noise level, diagnostic confidence, and lesion margin delineation, was conducted by two radiologists, with differences analyzed using the Kappa test. Results: Objective evaluation revealed a progressive decrease in lesion SD and a progressive increase in SNR and CNR from subgroups A1/B1 to A3/B3. The SD of Group A2 decreased by 57.4% compared to A4, while the SNR and CNR of A2 icreased by 19.3% and 24.6% compared to A4. Although subgroup B2 had a lower SNR than B5, the difference was not statistically significant. SNR and CNR in B2 increased by 24.1% and 11.9%, respectively, compared to B4. ERS gradually decreased from A1/B1 to A3/B3. ERS values in A2 and B2 increased by 27.0% and 39.4%, respectively, relative to A5 and B5. Although A3 had a lower ERS than A1 and A2, all DLIR subgroups exhibited higher ERS than A5; similar trends were observed in Group B. Subjective evaluation indicated good inter-reader agreement (Kappa > 0.61, p < 0.05). As DLIR strength increased, noise scores rose progressively in both groups. However, noise in A2 and B2 was lower than in A4/A5 and B4/B5. Diagnostic confidence and lesion margin delineation scores were highest in A2 and B2, while all subjective scores were lowest in A5 and B5. Discussion: Most prior studies evaluated the liver, vessels, or confirmed that image quality can be guaranteed at low doses. However, there are few studies on specific individual lesions. Therefore, this study aims to investigate specific individual lesions. The details and detection rate were analyzed separately to confirm the clinical acceptability of 2.5-mm DLIR image in different contrast lesions. Conclusion: For both high- and low-contrast hepatic lesions, DLIR provides superior image quality compared to ASIR-V, with the 2.5mm DLIR-M setting being optimal. DLIR-M reduces image noise, improves spatial resolution, and produces images more suitable for diagnostic purposes.

Background. Guided structured reporting has been proposed to address the limited availability of structured data in radiology, yet empirical evidence on its real-world adoption across users and imaging modalities remains scarce. Objective. To describe the adoption dynamics of a guided structured reporting system across multiple users and imaging modalities during a six-week implementation period. Methods. Retrospective observational study at two public tertiary hospitals in Abu Dhabi, United Arab Emirates. A guided structured reporting system was deployed for computed tomography (CT), magnetic resonance imaging (MRI), and mammography. Seven radiologists participated. The primary outcome was active in-software reporting time, recorded via system logs of mouse and keyboard interaction. Temporal trends in median reporting time per modality and individual user trajectories were analysed descriptively. After predefined data cleaning, 126 reports were included (84 CT, 27 MRI, 15 mammography). Results. Active in-software reporting time decreased across all modalities. Median reporting time fell from 130 s to 56 s for CT, from 383 s to 60 s for MRI, and from 126 s to 46 s for mammography (week 1 to week 6). Individual trajectories showed similar patterns, with the largest reductions during the early implementation phase. Subgroup analyses were limited by small sample sizes. Conclusions. Guided structured reporting was integrated into routine clinical workflows with temporal reductions in active reporting time across users and modalities, providing empirical evidence on the feasibility of workflow-integrated structured reporting in radiological practice.

Purpose: To develop and evaluate a flexible, software-defined radar platform for contactless, vendor-independent motion detection and correction in MRI. Methods: A continuous-wave (CW) Doppler radar was implemented using a software-defined radio and the open-source GNU Radio framework. The system was deployed inside a 1.5T MRI scanner and synchronized with MRI acquisitions. We evaluated the performance in a custom-developed internal motion phantom and in healthy volunteers to track respiration and bulk motion. The radar-derived signal was validated against cine MRI and used to demonstrate both retrospective and prospective motion management techniques in phantom and in healthy volunteers. Results: The radar provided robust motion signals that correlated strongly with image-based ground truth signals in both phantom and volunteer experiments. Signal characteristics were found to be frequency-dependent, enabling optimization for different motion regimes. Retrospective correction of free-breathing abdominal data using the radar signal effectively suppressed respiratory artifacts, achieving image quality comparable to a self-gating approach. Prospective triggering successfully reduced motion artifacts in the phantom study. The system also reliably detected sporadic events such as swallowing during neck imaging. Conclusion: Software-defined radar was demonstrated to be an effective platform for both prospective and retrospective motion correction. Its independence from the MRI system, ultra-wide band capabilities, and body-region versatility enable the adaptation of the technique for a wide range of imaging applications and protocols.

We present an ultrahigh-field magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize intact nontuberculous mycobacteria (NTM) at the molecular level. Hydrated and dried whole-cell Mycobacterium abscessus samples were investigated by combining conventional high-field ssNMR at 750 MHz with ultrahigh-field ssNMR at 1.2 GHz and ultrafast MAS at 100 kHz. To improve sensitivity and enable multidimensional experiments, 13C/15N isotope labeling was performed after growth in synthetic cystic fibrosis medium (SCFM). We utilized 1D 13C and multidimensional 1H-13C and 13C-13C ssNMR experiments to characterize the chemical composition, dynamics, and structural organization of the M. abscessus cell envelope. The isotope-labeling efficiency was found to be non-uniform across different molecular classes, with high incorporation into polysaccharides and lower incorporation into lipid and peptide-associated signals. INEPT- and CP-based experiments selectively probed flexible and rigid fractions of the samples, revealing substantial differences in linewidth, dynamics, and sensitivity between hydrated and dried preparations. Conventional 750 MHz experiments provided high-resolution multidimensional spectra and enabled identification of distinct chemical environments associated with peptidoglycan, arabinogalactan, mycolic acids, lipids, and peptide-associated components. Ultrahigh-field ssNMR at 1.2 GHz combined with ultrafast MAS and 1H detection substantially improved spectral resolution and sensitivity in particular per mg of sample amount, allowing detection of weak and previously unresolved resonances, including polysaccharide and possible nucleic-acid-associated signals. Together, these results demonstrate that ultra-high-field and ultrafast-MAS ssNMR enables detailed characterization of intact NTM cell envelopes under near-native conditions and provides a framework for future molecular investigations of antimicrobial interactions.