Objective: Emphysema is an important chronic obstructive pulmonary disease (COPD) phenotype characterized by the destruction of air spaces distal to the terminal bronchiole. Aiming to detect potential emphysema biomarkers and to assess the systemic effects of emphysema in blood plasma, we conducted a small cross-sectional shotgun proteomics study. Methods: This study included N = 40 participants divided into four subgroups (N = 10 per group): patients with emphysema and COPD (CE), patients with COPD but without emphysema (CN), healthy smokers (HS) and healthy never-smokers (HN). The participants were sampled non-probabilistically to be similar in terms of age, sex and comorbidities. Participants’ blood plasma was analyzed using liquid chromatography–mass spectrometry. Bioinformatic analysis included detection of differentially expressed proteins (DEPs) and overrepresentation analysis (ORA). Results: Across all groups, a total of 994 proteins were identified, with NADP-dependent malic enzyme (NADP-ME; encoded by ME1) being the only DEP in the CE vs. CN contrast. Proteins such as BMP1, ADAMTSL-2, -4 and IGFBP4, -5, 6 were identified to be upregulated in CE vs. HN. Fibulin-1, -3 and several immunoglobulin components were identified to be downregulated in the CE vs. HN contrast. ORA revealed several enriched processes, including serine-type endopeptidase activity, insulin-like growth factor I and II binding, and signaling receptor binding. Conclusion: We propose NADP-ME, an important enzyme of intermediary metabolism and redox homeostasis, as a potential biomarker candidate of emphysema. Notably, NADP-ME is also implicated in anoikis resistance. Additionally, changes in the expression levels of BMP1, ADAMTSL-2 and -4, and fibulin suggest potential major systemic effects of extracellular matrix perturbation. As all data was derived from LC-MS analysis, these findings need to be further evaluated with complementary methods.

Electrical pulse stimulation (EPS) represents a useful tool to study exercise‐related adaptations of muscle cells in vitro . Here, we examine the metabolic and secretory response of primary human muscle cells from metabolically healthy individuals to the EPS protocol reflecting the episodic nature of real‐life exercise training. This intermittent EPS protocol alternates high‐frequency stimulation periods with low‐frequency resting periods. Continuous EPS was used as a comparator. Radiometric assessment of glucose and fatty acid metabolism was complemented by examination of mitochondrial OxPHOS proteins, fiber‐type markers, and the release of selected myokines and extracellular vesicles into the media. Both EPS protocols facilitated glycogen synthesis and incomplete fatty acid oxidation (intermediary metabolites accumulation), while complete glucose and fatty acid oxidation (CO ₂ production) was increased only after the intermittent stimulation. Continuous stimulation elicited robust release of the contraction‐regulated myokines (IL6, IL8) into the media. Both EPS protocols increased expression of oxidative fiber‐type markers ( MYH2, MYH7 ), while inducing protein expression of a putative myokine, growth differentiation factor11 (GDF11) and a release of extracellular vesicles into the media. In conclusion, intermittent electrical pulse stimulation enhanced the rate of complete glucose and fatty acid oxidation in differentiated muscle cells from metabolically healthy individuals, while it was comparable to continuous stimulation in modulating markers of oxidative fibers and a putative myokine GDF11, and less effective in stimulating the release of myokines IL6, IL8, and extracellular vesicles into the media. Intermittent EPS—a protocol mimicking the episodic nature of exercise—can be used for studying metabolism and the secretome of skeletal muscle cells in vitro .

The pancreatic cancer-associated fibroblasts (pCAFs) are among the most active components of the pancreatic ductal adenocarcinoma (PDAC). The pCAFs being o

The extreme lethality and limited treatment options for pancreatic ductal adenocarcinoma (PDAC) underscore the urgent need for innovative therapeutic strategies. This study presents the first preclinical investigation of a cell-free gene-directed enzyme prodrug therapy (GDEPT) based on conditioned medium (CM) from placenta-derived mesenchymal stem cells (PlacMSCs) engineered to express the yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT) fusion enzyme. The CM was concentrated tenfold (cCM) and characterized by proteomics, transmission electron microscopy, and Western blotting, confirming extracellular vesicle (EV) enrichment and UPRT expression. Therapeutic efficacy was evaluated in coculture models comprising PDAC cell lines (BxPC-3, MIA PaCa-2, SU.86.86), patient-derived xenograft organoids (PDXOs), and cancer-associated fibroblasts (PCAFs). Immunocytochemistry and Western blot analyses revealed a predominant myofibroblastic CAF phenotype, characterized by strong alpha-smooth muscle actin (αSMA) expression and low interleukin-6 levels. Treatment with yCD::UPRT-PlacMSC-cCM in the presence of 5-fluorocytosine (5-FC) enabled efficient enzymatic conversion to 5-fluorouracil (5-FU), yielding 10 μg/mL from an initial 100 μg/mL of 5-FC. This resulted in robust, dose-dependent cytotoxicity (50 % to 80 % reduction in viability) across monocultures and stromal-rich cocultures, effectively overcoming PCAF-mediated drug resistance. Therapeutic response was governed primarily by tumor cell characteristics rather than PCAF heterogeneity. In PDXOs derived from two early-stage (IA, IIB) primary tumors and one metastatic lesion, 100 µL of yCD::UPRT-PlacMSC-cCM induced cytotoxicity comparable to 1 μg/mL of 5-FU, while 25 µL was insufficient to significantly reduce viability. Collectively, these findings demonstrate that yCD::UPRT-PlacMSC-cCM delivers potent, stromal-bypassing cytotoxicity in PDAC models and represents a promising cell-free therapeutic approach for this treatment-refractory cancer.

Background: Systemic sclerosis (SSc)-associated interstitial lung disease (SSc-ILD) is the leading cause of morbidity and mortality in patients with SSc, with an unmet need for validated, minimally invasive biomarkers for early detection. Extracellular vesicles (EVs) present underexplored pathogenic players and potential biomarker sources in SSc-ILD. We performed a proteomic shotgun study aiming to identify disease-specific protein signatures and potential biomarker candidates. Methods: The study included 30 SSc patients divided into SSc-ILD and SSc w/o ILD groups and 20 matched controls. Plasma-derived EV-enriched fractions were analysed using liquid chromatography–mass spectrometry. Bioinformatic analysis, including differentially expressed proteins (DEPs), functional enrichment, protein–protein interaction network and Markov Cluster (MCL) analysis was performed. Results: Analysis of DEPs showed 14 significantly upregulated and 1 downregulated protein when comparing the SSc-ILD to the SSc w/o ILD group, 222 upregulated and 257 downregulated proteins between the SSc-ILD and control group, as well as 362 upregulated and 492 downregulated proteins between the SSc w/o ILD and control group. Functional enrichment analysis and MCL analysis pointed to disease-specific processes of extracellular matrix (ECM) and immune dysregulation, which largely overlapped between SSc-ILD and SSc w/o ILD groups. Among identified DEPs, SP-B, Cav-1 and Siglec-5 emerged as potential candidate biomarkers for SSc-ILD. Conclusions: Proteomic analysis of plasma-derived EV-enriched fractions shows potential EV involvement in pathogenic SSc processes, mainly related to ECM and immune dysregulation, as well as potential candidate biomarkers for SSc-ILD. Further studies are required to validate these results and assess biomarker potential and translational applicability of identified proteins.

Soluble proteins present in spent culture media (SCM) have been proposed as potential indicators of embryo viability. However, results across studies remain inconsistent, and the identity of reliable protein biomarkers is still unclear. To evaluate the potential of secretome profiling for non-invasive embryo assessment, we applied state-of-the-art mass spectrometry to analyze the protein composition of SCM from individual successful and unsuccessful human embryo cultures, alongside corresponding controls cultured under identical conditions. Surprisingly, both untargeted (nanoLC-TIMS-TOF-MS/MS) and targeted (UHPLC-QqQ-MS/MS) proteomic analyses consistently detected multiple human proteins in unconditioned media, indicating that these signals are not of embryonic origin. Undeclared proteins were also identified in media sampled before their first use, with considerable variability observed across production batches. Supplementation experiments demonstrated that this variability originates from the addition of plasma-derived human serum albumin, which introduces a range of contaminating proteins. In contrast, recombinant albumin did not contribute detectable serum proteins. These findings indicate that the protein background in commercial IVF media is an overlooked variable, not only compromising the reproducibility and interpretation of secretome analyses in research, but also contributing to unstandardized culture conditions in clinical IVF practice. While this study evaluated 13 production lots of monophasic media from two different manufacturers, the results underscore a broader need for transparency and standardization in IVF media composition. The adoption of fully chemically defined IVF media, free from purified serum components, would support more consistent embryo culture conditions and advance the discovery of biomarkers in SCM.