(1) Background: Sepsis is characterized by profound heterogeneity of immune responses, complicating biomarker-based prediction of clinical outcomes. Latent human cytomegalovirus (HCMV) infection is one of the strongest modulators of the human immune system and may influence cytokine-mediated signaling during sepsis. (2) Methods: In this post hoc analysis of 331 patients from the prospective multicenter SepsisDataNet.NRW cohort (German Clinical Trial Registry No. DRKS00018871), we quantified 13 serum cytokines on day 1 after sepsis diagnosis and determined HCMV IgG serostatus via ELISA. Using nested cross-validated logistic regression with exhaustive feature selection, we identified cytokine panels predictive of 30-day survival in the total cohort and in subgroups stratified by HCMV serostatus. (3) Results: In the total cohort, a four-cytokine panel (IL-6, IL-10, TNF-α, IL-12p70) predicted 30-day survival with a cross-validated area under the curve (AUC) of 0.66 [95% CI: 0.59–0.72]. Stratification by HCMV serostatus revealed distinct predictive profiles: in HCMV-seropositive patients, a two-cytokine model (IL-10, IL-23) achieved an AUC of 0.69 [95% CI: 0.61–0.77], whereas in seronegative patients, a model based on IL-8 and IL-17A failed to generalize (AUC = 0.47 [95% CI: 0.33–0.61]). Kaplan–Meier analysis confirmed a significant separation of survival curves for the HCMV-seropositive group (p < 0.001) but not for seronegative patients (p = 0.282). (4) Conclusions: HCMV serostatus defines an immunological context in which cytokine-based prediction of sepsis outcome becomes feasible. These data suggest that viral serostatus should be systematically incorporated into biomarker discovery and immunophenotyping approaches to improve the reproducibility and biological interpretability of sepsis endotyping.

Sepsis is a life-threatening condition characterized by a dysregulated immune response to infection. Toll-like receptor 4 plays a central role in pathogen recognition and inflammatory signalling and has been considered a key driver of sepsis pathophysiology. Pharmacological inhibition of this receptor showed beneficial effects in experimental models but failed in clinical trials. We therefore aimed to quantify in vivo activation of Toll-like receptor 4 in patients with sepsis and to determine its association with 30-day survival. Peripheral blood mononuclear cells were obtained from 100 patients with sepsis enrolled in the SepsisDataNet.NRW cohort. Samples were collected on day 1 (within 36 h after diagnosis) and day 4. Activation of TLR4 was quantified by measuring receptor phosphorylation using a validated proximity ligation assay. Survival analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression models to assess the association between receptor activation and 30-day mortality. Overall activation of TLR4 was low, with median values below one signal per cell at both day 1 and day 4. Despite the generally low levels, a subgroup of patients showed increased receptor activation. Higher activation was associated with significantly reduced 30-day survival. Patients with elevated activation had a higher risk of death both at day 1 (HR 2.03, 95

Mineralized bone tissue is essential for skeletal integrity, mechanical stability, and mineral homeostasis. Osteocytes, the most abundant bone cell type, play central roles in bone remodeling and endocrine regulation but remain difficult to investigate because of their embedded localization within the mineralized matrix. Consequently, the molecular mechanisms underlying osteoblast-to-osteocyte differentiation remain incompletely understood.Here, we performed a comprehensive quantitative proteomic analysis of the widely used human SaOS-2 cell line during osteoblast-to-osteocyte-like differentiation. More than 5,000 proteins were quantified, providing a comprehensive proteomic roadmap of this differentiation process. Proteomic profiling demonstrated extensive molecular remodeling accompanied by extracellular matrix reorganization and dynamic changes in collagen abundance. Although differentiated SaOS-2 cells retained characteristics of late osteoblasts and did not fully recapitulate the phenotype of mature osteocytes, they acquired a distinct osteocyte-like proteomic signature. Furthermore, we identified and orthogonally validated several candidate protein markers associated with the osteoblast and osteocyte-like states.Together, our study provides the first comprehensive protein-level characterization of SaOS-2 osteoblast-to-osteocyte-like differentiation. Beyond improving the molecular characterization of this widely used model, it provides a valuable resource for future studies investigating bone biology, extracellular matrix remodeling, and skeletal disease

The chicken embryo is a well-established model for studying vertebrate heart development; however, comprehensive proteomic analyses of cardiac maturation in this system are lacking. Here, we present a quantitative, data-independent acquisition mass spectrometry-based proteomic analysis of embryonic chicken hearts at three key developmental stages (E4, E8, and E10). Across these stages, we quantified more than 5,500 proteins and resolved distinct temporal expression trajectories corresponding to early biosynthetic programs, mid-embryonic morphogenetic remodeling, and late-stage metabolic and contractile maturation. Our analysis uncovered stage-specific abundance patterns of established cardiac lineage markers and identified previously uncharacterized proteins with distinct temporal dynamics, suggesting potential roles in specific phases of heart development. The resulting temporal proteomic atlas provides a foundational resource for integrating proteomic and transcriptomic data and for investigating molecular mechanisms underlying cardiac morphogenesis and congenital heart disease. Data are available via ProteomeXchange with identifier PXD076525.

Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are key microbial metabolites that play crucial roles in modulating host–microbiome interactions, immune function, and metabolic homeostasis. Due to their significant involvement in both physiological and pathological processes, there is increasing demand for reliable, sensitive, and high-throughput analytical methods to quantify SCFAs in biological matrices. Liquid biopsies, such as plasma and serum, offer a minimally invasive means to monitor SCFA levels, making them valuable for both research and clinical applications. In this study, we introduce a workflow for the quantitative profiling of acetate, propionate, and butyrate using direct infusion mass spectrometry (DI-MS). The method employs a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer coupled with the TriVersa NanoMate™ robotic nanoflow ion for automated sample introduction. To improve ionization efficiency and detection sensitivity, SCFAs were extracted with isopropanol (IPA) and chemically derivatized prior to analysis. Method validation included determination of limits of detection (LOD), limits of quantification (LOQ), linearity, reproducibility, and assessment of matrix effects. The method was further applied to both plasma and serum samples to demonstrate its utility in clinically relevant matrices. The developed DI-MS-method demonstrated good analytical performance, including low limits of detection (LOD) for all three SCFAs − 0.1 µM for acetate, 0.01 µM for propionate, and 0.05 µM for butyrate - alongside wide linear dynamic ranges (r² ≥ 0.991). The assay showed high reproducibility, with low within-run (CV < 15

Pulmonary arterial hypertension (PAH) is a serious disorder, in which increased vascular tone is one of the critical hallmarks. Since beta arrestins (bArrs) have been shown to regulate smooth muscle tone in the airways, we investigated the function of bArr1 in the pulmonary vasculature. Here, we report that bArr1 is essential for maintaining normal pulmonary arterial tone. Specifically, pulmonary arteries from bArr1-/- mice exhibited reduced NO-dependent vasorelaxation due to impaired soluble guanylyl cyclase (sGC) activity, which was restored by the heme-independent sGC activator BAY58-2667. We identified bArr1 as a binding partner of sGC and the sGC heme reductase cytochrome b5 reductase (Cyb5r3), indicating that bArr1 is vital for sensitizing sGC to NO. Finally, mice with either ubiquitous or smooth muscle-specific bArr1 deficiency developed pulmonary hypertension (PH). These findings highlight the important role of bArr1 in regulating pulmonary vascular tone and propose it as a potential therapeutic target for the treatment of PH.

BackgroundSepsis, a life-threatening condition caused by a dysregulated host response to infection, remains a major cause of mortality worldwide. Identifying reliable biomarkers for prognosis and treatment is urgently needed. This study investigates the role of the Apoptosis Inhibitor of Macrophages (AIM), also known as CD5L, as a potential prognostic biomarker and therapeutic target in sepsis.MethodsWe measured free and total AIM concentrations in 90 septic patients enrolled in SepsisDataNet.NRW cohort (German Clinical Trial Registry No. DRKS00018871; http://www.sepsisdatanet.nrw). Blood samples were collected on days 1, 4, and 8, and AIM levels were quantified using ELISA. Kaplan-Meier analysis and Cox regression were performed to assess the association between AIM levels and 30-day survival. Western blot analysis was performed to detect AIM in human serum IgM and in the IgM-enriched intravenous immunoglobulin IVIG preparation Pentaglobin®.ResultsHigh total AIM concentrations (>85 ng/ml) were significantly associated with improved 30-day survival on day 1 (HR: 3.131, 95% CI: 1.629-6.019, p = 0.009), 4 (HR: 2.525, 95% CI: 1.198-5.322, p = 0.0042), and day 8 (HR: 2.317, 95% CI: 0.8565-6.266, p = 0.0457). Free AIM showed a significant association with survival only on day 8 (HR: 2.374, 95% CI: 0.8721-6.461, p = 0.0393).ConclusionTotal AIM concentration is a significant predictor of a 30-day survival in sepsis, supporting its potential use as a prognostic biomarker. Our findings also suggest that AIM may serve as a valuable prognostic biomarker and a potential target for immune-modulating therapies, including IgM-enriched intravenous immunoglobulins (IVIGs).

Protein inference is an often neglected though crucial step in most proteomic experiments. In the bottom-up proteomic approach, the actual molecules of interest, the proteins, are digested into peptides before measurement on a mass spectrometer. This approach introduces a loss of information: The actual proteins must be inferred based on the identified peptides. While this might seem trivial, there are certain problems, one of the biggest being the presence of peptides that are shared among proteins. These amino acid sequences can, based on the database used for identification, belong to more than one protein. If such peptides are identified in a sample, it cannot be said which proteins actually were in the sample, but only an estimate on the most probable proteins or protein groups can be given based on a predefined inference strategy.Here we describe the effect of the chosen database for peptide identification on the number of shared peptides. Afterward, the mainly used protein inference methods will be sketched, and the necessity of stringent false discovery rate on peptide and protein level is discussed. Finally, we explain how the tool "PIA or protein inference algorithms" can be used together with the workflow environment KNIME and OpenMS to perform protein inference in a common proteomic experiment.

The SH-SY5Y cell line is a triple-cloned subline of SK-N-SH cells originally isolated in the early 1970s from a bone marrow biopsy of a four-year-old female patient suffering from neuroblastoma. Since then, this cell line has been used as one of the major cell culture models in neuroscience and to study neurodegeneration, as it comprises many of the biochemical and functional properties of neural precursor cells. Differentiation of neuronal precursor cells into a more mature phenotype represents one of the key steps and directed differentiation utilising various reagents is thought to provoke a defined neuronal subtype. Unfortunately, until now there is no consensus, which protocol shall be utilised to reach a specific neuronal subtype. Thus, the aim of the present work was to evaluate four common standard protocols for the differentiation of SH-SY5Y cells and to investigate the respective influences of varying parameters of these differentiation strategies. For this purpose, morphological analyses, mass spectrometry-based quantification of specific marker proteins, time-course protein expression profiling and global proteomics were conducted. On the level of morphology a low serum concentration favoured the abundance of mature neuronal cells containing long and branched neurites. Further low serum levels favoured the expression of dopaminergic marker proteins, in particular DDC, especially when utilising retinoic acid as differentiation agent. Our study clearly shows that an a priori characterisation of SH-SY5Y cells is indispensable to assess the abundance of neuronal subtypes and by that to ensure that the utilised differentiation approach is appropriately aligned with the specific research question.

Background: Short-chain fatty acids (SCFAs), including propionic acid (PA), are key in immunological research. Supplementing PA has shown benefits for autoimmune diseases. A comprehensive understanding of the PA pharmacokinetics is essential for the optimal design and execution of studies utilizing orally administered PA. Objective: We propose two methods of measuring PA in serum, carried out by different laboratories. Design: Blood samples from 20 volunteers were collected hourly following PA supplementation. Methods: Serum propionate quantification was performed with two independent mass spectrometry-based (MS) analyses, including liquid-chromatography (LC)-MS and direct-infusion (DI)-MS. Results: PA levels increased within 1 h of ingestion of 500 mg PA. Serum concentrations ranged from 1.3 to 4.5 µmol/L, rising significantly after 1 h ( p < 0.05). Serum levels returned to baseline within 2 h. No significant differences were found regarding sex or diet. Conclusion: The shown pharmacokinetics can be used in future PA research.

Background: Duchenne muscular dystrophy (DMD), which affects 1 in 3500 to 5000 newborn boys worldwide, is characterized by progressive skeletal muscle weakness and degeneration. The reduced muscle regeneration capacity presented by patients is associated with increased fibrosis. Satellite cells (SCs) are skeletal muscle stem cells that play an important role in adult muscle maintenance and regeneration. The absence or mutation of dystrophin in DMD is hypothesized to impair SC asymmetric division, leading to cell cycle arrest. Methods: To overcome the limited availability of biopsies from DMD patients, we used our 3D skeletal muscle organoid (SMO) system, which delivers a stable population of myogenic progenitors (MPs) in dormant, activated, and committed stages, to perform SMO cultures using three DMD patient-derived iPSC lines. Results: The results of scRNA-seq analysis of three DMD SMO cultures versus two healthy, non-isogenic, SMO cultures indicate reduced MP populations with constant activation and differentiation, trending toward embryonic and immature myotubes. Mapping our data onto the human myogenic reference atlas, together with primary SC scRNA-seq data, indicated a more immature developmental stage of DMD organoid-derived MPs. DMD fibro-adipogenic progenitors (FAPs) appear to be activated in SMOs. Conclusions: Our organoid system provides a promising model for studying muscular dystrophies in vitro, especially in the case of early developmental onset, and a methodology for overcoming the bottleneck of limited patient material for skeletal muscle disease modeling.

Proteomics of laser-dissected lipofuscin from aged, healthy brains reveals Palmitoyl-Protein Thioesterase 1 (PPT1) and other CLN proteins as constituents. PPT1 is increasingly sequestered to lipofuscin during ageing. Protein sequestering into lipofuscin may contribute to physiological neuronal ageing.

The β-subunit (Cavβ) is a central component of the voltage-gated calcium channel complex. It lacks transmembrane domains and exhibits both channel-related and non-related functions. Previous studies have shown that, in the absence of the Cavα1 pore-forming subunit, electrostatic interactions between the N-terminus of Cavβ2e and the plasma membrane mediate its anchoring to the cell surface. Here, we demonstrate that, upon phospholipase C activation, Cavβ2e dissociates from the plasma membrane and homogeneously distributes between the cytosol and the nucleus. Mutagenesis analysis identified critical residues in the N-terminus of the protein, including a stretch of positively charged amino acids and a dileucine motif, which serve as nuclear import and export signals, respectively. Fusion of the Cavβ2e N-terminus to a trimeric YFP chimeric construct shows that this segment suffices for nuclear shuttling. Thus, the N-terminus of Cavβ2e emerges as a regulatory hotspot region controlling the subcellular localization of the protein. Quantitative mass spectrometry analysis revealed that the heterologous expression of a nuclear-enriched Cavβ2e mutant regulates gene expression. Our findings demonstrate the presence of active nuclear localization signals in Cavβ2e that enables its nuclear targeting and regulation of protein expression. Furthermore, they establish the membrane-associated Cavβ2e as a novel signaling mediator within the phospholipase C cascade.

INTRODUCTION/AIMS:Desminopathies are a group of rare human myopathies and cardiomyopathies caused by pathogenic variants of the desmin gene. Here, we analyzed the effects of the R349P mutant desmin on the proteomic profiles of individual fiber types of murine skeletal muscle. METHODS:Soleus and tibialis anterior muscles from hetero- and homozygous R349P desmin knock-in mice and wild-type siblings were used to collect fiber type-specific material by laser microdissection to determine their proteomic profiles. RESULTS:Aberrant proteomic profiles were observed in all four fiber types of homozygous mice. Type I and IIa fibers from homozygous muscle showed an increased abundance of 15 fibrotic proteins, for example, collagens I, IV, and VI, and associated proteins. Filamin-C, xin actin-binding repeat-containing proteins 1 and 2, and Kelch-like protein 41 were more abundant in homozygous fibers. A high number of proteins associated with the mitochondrial complexes had markedly lower amounts in all types of homozygous and type IIb heterozygous fibers, whereby 20 proteins of complex I, 6 proteins of complex III, 7 proteins of complex IV, and 4 proteins of complex V were found to be decreased in homozygous mice in at least one fiber type. This reduction included all mtDNA-encoded proteins of complexes I and V, as well as ADP/ATP translocase 1 and 2. DISCUSSION:Our proteomic findings highlight a more severe myodegenerative process in fibers derived from homozygous R349P desmin knock-in mice. R349P desmin altered the abundance of proteins of the sarcomeric and extrasarcomeric cytoskeleton, extracellular matrix, and mitochondrial energy metabolism.

Comprehensive characterization of platelets requires various functional assays and analysis techniques, including omics-disciplines, each requiring an individual aliquot of a given sample. Consequently, the sample material per assay is often highly limited rendering downscaling a prerequisite for effective sample exploitation. Here we present a transfer of our recently introduced 96-well-based proteomics workflow (PF96) into the 384-well format (PF384) allowing for a significant increase in sensitivity when processing minute platelet protein amounts. In addition, the 4-fold higher throughput (1500 samples per lab worker per week) allows to easily meet the throughput capacities of modern LC-MS instruments. We determined optimal sample loads followed by highlighting the strengths in comparison to our previous sample preparation approach by processing only 3 µg of purified platelet protein from 22 healthy donors. Major advantages are: (I) improved identification and analyte recovery, especially of low copy number proteins, with signal intensity gains of +130 % and +107 % (peptide and protein level, respectively) (II) substantial intensity gains for key-players in platelet activation including the membrane receptors PAR4, P2X1, GPVI, GPV, GPIX and the downstream mediators AKT, PKA, Rap1, Lyn (III) improved reproducibility with a reduction of technical variance from 22 / 25 % down to 16 / 19 % for detection of lower / higher abundant disease markers and (IV) a 4-fold increase in sample preparation throughput. Taken together, these advantages render PF384 a promising future in clinical proteomics and might pave the way of platelet proteomics with minute sample amounts into molecular diagnostics.

Tauopathies are characterized by the progressive accumulation of abnormal tau species, which disrupt the autophagy-lysosomal pathway (ALP), a critical system for degrading intracellular macromolecules and aggregated proteins, causing toxicity and cell death. This study investigates the impact of the N-terminally truncated Tau35 protein overexpression on proteolytic pathways, including effects on autophagy and endo-lysosomal processes. Using a Tau35 mouse model and SH-SY5Y cell lines stably expressing either the Tau35 fragment or full-length tau, we employed western blotting, proteomic analysis of lysosome-enriched brain fractions, proteolysis/endocytosis assays, and live-cell imaging with the lysotracker reporter to assess protein degradation and lysosomal function. Our findings identify early pathological changes in endo-lysosomal processes, including increased endocytosis, proteolytic dysfunction and lysosomal motility abnormalities, associated with Tau35-induced toxicity. This work extends previous research by providing new insights into the mechanisms of Tau35-induced neurotoxicity, offering a foundation for developing targeted therapeutic strategies to address tauopathies. ### Competing Interest Statement The authors declare no competing interests. Graham Fraser is an employee of AstraZeneca plc.

Comprehensive characterization of platelets requires various functional assays and analytical techniques, including omics disciplines, each demanding a separate aliquot of the given sample. Consequently, sample material for each assay is often highly limited, necessitating the downscaling of methods to work with just a few micrograms of platelet protein.Here, we present a novel sample preparation platform for proteomics analysis using only 3 μg of purified platelet protein, corresponding to 2 × 106 platelets, which can be obtained from approximately 2 to 8 μL of blood from a healthy individual (1.5 × 105-4.5 × 105 platelets/μL) or approximately 100 μL of blood from a patient with severe thrombocytopenia (<2 × 104 platelets/µL).Using this platform, we detected a significant fraction of key players in the platelet activation cascade and, most importantly, identified 36 clinically relevant platelet disease markers even with a non-state-of-the art instrument. This makes LC-MS-based proteomics a highly attractive alternative to conventional assays, which often require milliliters of blood. Our platform transitions from our previously established 96-well proteomics workflow (PF96), which has been successfully employed in numerous platelet proteomics studies, into the 384-well format. This transition is accompanied by (1) a more than two-fold increase in sensitivity, (2) improved reproducibility, (3) a four-fold increase in throughput, allowing 1,536 samples to be processed per lab worker per week, and (4) reduced sample preparation costs.Thus, LC-MS-based platelet proteomics offers a compelling alternative to immunoaffinity assays (which depend on antibody availability and quality), as well as to genomic assays (which can only reveal genotypes). In summary, in conjunction with recent advances in LC-MS instrumentation, our platform represents a highly valuable tool for rapid phenotyping of platelets in research with extraordinary potential for future employment in companion or routine diagnostics.

The experience of adversity in childhood can have life-long consequences on health outcomes. In search of mediators of this relationship, alterations of bio-behavioral and cellular regulatory systems came into focus, including those dealing with basic gene regulatory processes. System biology oriented approaches have been proposed to gain a more comprehensive understanding of the complex multiple interrelations between and within layers of analysis. Here, we used co-expression based, supervised and unsupervised single and multi-omics systems approaches to investigate the association between childhood adversity and gene expression, protein expression and DNA methylation in CD14+ monocytes in the context of psychosocial stress exposure, in a sample of healthy adults with (n = 29) or without (n = 27) a history of childhood adversity. Childhood adversity explained some variance at the single analyte level and within gene and protein co-expression structures. A single-omics, post-stress gene expression model differentiated best between participants with a history of childhood adversity and control participants in supervised analyses. In unsupervised analyses, a multi-omics based model showed best performance but separated participants based on sex only. Multi-omics analyses are a promising concept but might yield different results based on the specific approach taken and the omics-datasets supplied. We found that stress associated gene-expression pattern were most strongly associated with childhood adversity, and integrating multiple cellular layers did not results in better discriminatory performance in our rather small sample. The capacity and yield of different omics-profiling methods might currently limit the full potential of integrative approaches.