Search Results (399)

Background: Intervertebral disc degeneration (IVDD) is closely linked to low back pain (LBP), a leading cause of disability worldwide. IVDD is characterized by the loss of proteoglycans (PGs), extracellular matrix (ECM) degradation, and reduced hydration of the nucleus pulposus (NP). Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) exhibit tissue repair and immunomodulatory effects and are emerging as promising cell-free therapeutics. Methods: We established a rat IVDD model via fluoroscopy-guided needle puncture of three consecutive coccygeal discs and confirmed degeneration through Alcian Blue and hematoxylin & eosin (H&E) staining. The gene expression of inflammatory and pain markers (ADRβ2, COMP, CXCL1, COX2, PPTA, MMP13, YKL40) was measured by qPCR. Subsequently, we implanted hUC-MSCs or EVs to evaluate their reparative potential. Results: Upregulation of inflammatory and pain genes in IVDD was associated with an immunomodulatory response. Tracking DiI-labelled hUC-MSCs and EVs revealed enhanced survival of hUC-MSCs, retention of EVs, and dispersion within rat tail discs; EVs showed greater retention than hUC-MSCs. Implanted EVs were internalized by NP cells and remained within degenerative IVDs. EVs passively diffused, accumulated at the injury site, interacted with host cells, and enhanced function, as shown by increased expression of human chondrocyte-related markers (SOX9, TGFβ1, TGFβ2, COL2) compared to hUC-MSC treatment. Histological analysis of two weeks post-transplantation showed NP cellular patterns resembling chondromas in treated discs. EVs integrated into and distributed within degenerated NP regions, with greater glycosaminoglycan (GAG) content. Conclusions: Overall, hUC-MSC EVs demonstrated superior regenerative capacity, supporting a safe, cell-free strategy for disc repair. Full article

We evaluated the potential of Fourier-transform infrared (FTIR) microspectroscopy for non-invasive biochemical profiling of rhesus macaque embryonic stem cells (rhESCs) cultured in either conventional FGF2/KOSR medium or a novel formulation, ALGöX. Cells from both conditions were analyzed by immunocytochemistry, RNA sequencing, and high-resolution FTIR profiling. Molecular marker expression patterns and transcriptional profiles revealed that rhESCs maintained in FGF2/KOSR were in the primed pluripotent state, whereas those cultured in ALGöX adopted a naïve-like state. FTIR spectra showed consistent differences in protein, lipid, and nucleic acid signatures, with ALGöX-cultured cells displaying higher amide I/II and nucleic acid absorbance and FGF2/KOSR-cultured cells exhibiting stronger lipid-associated bands. Principal component analysis (PCA) separated the two groups along PC−1 (64% variance), and partial least squares discriminant analysis (PLS-DA) classified samples with 100% specificity and 100% sensitivity. These findings demonstrate that FTIR microspectroscopy can reliably discriminate pluripotent state–specific biochemical features in non-human primate PSCs, providing a rapid and label-free approach for monitoring stem cell identity and quality. Full article

Progress in clinical diagnosis increasingly relies on innovative technologies and advanced disease biomarker detection methods. While cell labeling remains a well-established technique, label-free approaches offer significant advantages, including reduced workload, minimal sample damage, cost-effectiveness, and simplified chip integration. These approaches focus on the morpho-biophysical properties of cells, eliminating the need for labeling and thus reducing false results while enhancing data reliability and reproducibility. Current label-free methods span conventional and advanced technologies, including phase-contrast microscopy, holographic microscopy, varied cytometries, microfluidics, dynamic light scattering, atomic force microscopy, and electrical impedance spectroscopy. Their integration with artificial intelligence further enhances their utility, enabling rapid, non-invasive cell identification, dynamic cellular interaction monitoring, and electro-mechanical and morphological cue analysis, making them particularly valuable for cancer diagnostics, monitoring, and prognosis. This review compiles recent label-free cancer cell detection developments within clinical and biotechnological laboratory contexts, emphasizing biophysical alterations pertinent to liquid biopsy applications. It highlights interdisciplinary innovations that allow the characterization and potential identification of cancer cells without labeling. Furthermore, a comparative analysis addresses throughput, resolution, and detection capabilities, thereby guiding their effective deployment in biomedical research and clinical oncology settings. Full article

In human cells, the biogenesis of membrane proteins, which account for one quarter of polypeptides and sixty percent of human drug targets, is initiated at the membrane of the endoplasmic reticulum (ER). This process involves N-terminal signal peptides or transmembrane helices in the membrane protein precursors. Over one hundred proteins enable membrane-targeting and -insertion of the precursors as well as their folding and covalent modifications. Four targeting pathways to the Sec61 channel in the ER membrane with their effectors and three cooperating or independent membrane protein–insertases have been identified. We combined knock-down of individual components of these pathways and insertases in HeLa cells with label-free quantitative mass spectrometric analysis of the proteomes. Differential protein abundance analysis in comparison to control cells was employed to identify clients of components involved in the targeting or membrane insertion of precursors. Alternatively, knock-out cells or relevant patient fibroblasts were employed. The features of the client polypeptides were characterized to identify the client types of the different components and, ideally, their rules of engagement. In this review/article-hybrid, the focus is on global lessons from and limitations of the proteomic approach in answering the cell biological question, as well as on new aspects, such as N-terminal acetylation of membrane protein precursors. Full article

Background/Objectives: Mass-forming chronic pancreatitis (MFP) and pancreatic ductal adenocarcinoma (PDAC) can present with overlapping radiological, clinical, and serological features in patients with underlying chronic pancreatitis (CP), making differential diagnosis particularly challenging. Current diagnostic tools, including CA19-9 and endoscopic ultrasound (EUS) imaging, often lack the specificity needed to reliably distinguish between these conditions. The objective of this study was to investigate whether the proteomic profiling of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) samples could provide molecular-level discrimination between MFP and PDAC in patients with CP. Methods: Thirty CP patients with solid pancreatic lesions were prospectively enrolled: 15 with histologically confirmed PDAC and 15 with MFP. Traditional diagnostic parameters, including CA19-9 levels and EUS characteristics, were recorded but found insufficient for differentiation. EUS-FNA samples were analyzed using label-free mass spectrometry. A total of 928 proteins were identified in PDAC samples and 555 in MFP samples. Differential abundance analysis and pathway enrichment were performed. Results: Overall, 88 proteins showed significant differential abundance between PDAC and MFP samples, of which 26 met stringent statistical thresholds. Among these, Carboxylesterase 2 (CES2), Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (CEACAM1), Lumican (LUM), Transmembrane Protein 205 (TMEM205), and NAD(P)H Quinone Dehydrogenase 1 (NQO1) emerged as key discriminatory proteins. Pathway enrichment analysis revealed distinct biological processes between the groups, including mitochondrial fatty acid β-oxidation, Rho GTPase signaling, and platelet degranulation. Conclusions: Proteomic signatures derived from EUS-FNA samples offer a promising molecular approach to distinguish inflammatory pseudotumoral lesions from malignant pancreatic tumors in CP patients. This minimally invasive strategy could enhance diagnostic accuracy where current methods fall short. Further validation in larger, multicenter cohorts is warranted to confirm these findings and evaluate their clinical applicability. Full article

The search for natural alternatives to sodium nitrite in meat products is driven by concerns about consumer health and the need to maintain product quality and safety. In this study, the effect of sodium nitrite reduction on the quality parameters of canned pork meat with 1% lyophilized cell-free supernatant (CFS) from L. paracasei B1, during 30 days of storage, was assessed. Reduction of sodium nitrite content led to measurable changes in the color, texture, and oxidative stability of canned pork; however, the presence of 1% CFS helped preserve color, alleviated the negative impact on textural parameters, and limited lipid oxidation, thereby counteracting the typical consequences of nitrite reduction. Among the tested variants, S_75, containing 75% of the standard nitrite dose, showed the best overall balance between color retention, textural integrity, and oxidative stability. Samples without nitrite (S_0) exhibited a noticeable increase in lightness (L*) and decrease in redness (a*) over time, accompanied by a shift towards yellow-brown hues (b*, C*, H°). Importantly, the total color difference (ΔE) was least pronounced in the S_75 variant, with values of approximately 2.5 after 1 day and 2.7 after 30 days, which was markedly lower than in S_50 (ΔE ≈ 6.0 and 3.9) and S_0 (ΔE ≈ 7.9 and 8.5), thereby confirming superior color retention and overall stability during storage. Texture analysis showed that initial hardness and chewiness were higher in nitrite-free samples (S_0), suggesting that the complete omission of nitrite may negatively affect product structure. Nevertheless, all variants softened during storage, and samples with higher nitrite content, particularly S_75, retained better elasticity and cohesiveness. Lipid oxidation, expressed as TBARS values, progressed fastest in samples completely depleted of nitrite (S_0), increasing from 0.31 mg MDA/kg (day 1) to 1.35 mg MDA/kg (day 30), which confirms the antioxidant role of sodium nitrite. Interestingly, the presence of 1% CFS in the variants with reduced nitrite content partially mitigated this effect, as TBARS values in S_75 increased only from 0.29 to 0.46 mg MDA/kg, and, in S_50, from 0.45 to 0.66 mg MDA/kg, compared to the nitrite-free variant. This suggests that CFS may also have contributed to antioxidant protection. Fatty acid profiles remained relatively consistent across methods. Microbiological analysis revealed no significant differences between groups. These results demonstrate that partial nitrite reduction combined with CFS is effective, highlighting the potential of CFS as a promising functional additive in clean label meat preservation. Furthermore, reducing the sodium nitrite content in canned pork products may contribute to improved consumer health by reducing exposure to potentially harmful nitrosamine precursors. Full article

Acute lymphoblastic leukemia (ALL) represents the most common type of cancer in the pediatric population. The (1;19)(q23;p13) translocation is a primary chromosomal abnormality present in 3–12% of ALL cases. The current study aims to develop a label-free innovative nanodevice for the ultrasensitive diagnosis of the TCF3-PBX1 chimeric oncogene, featuring simplified operation and rapid analysis using minimal sample volumes, which positions it as a superior alternative for clinical diagnostics and early leukemia identification. The biosensor system was engineered on a nanostructured platform composed of polypyrrole (PPy) and a novel chemically functionalized hybrid nanocomposite of platinum nanospheres and titanium dioxide nanoparticles (TiO₂@Pt). Single-stranded oligonucleotide sequences were chemically immobilized on the nanoengineered transducer to enable biospecific detection. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), ultraviolet-visible spectroscopy (UV-Vis), and atomic force microscopy (AFM) were used to characterize each stage of the biotechnological device fabrication process. The analytical properties of the sensing tool were explored using recombinant plasmids containing the TCF3-PBX1 oncogenic sequence and clinical specimens from pediatric patients with B-cell ALL. After exposing the molecular monitoring system to the genetic target, significant variations were observed in the voltammetric oxidation current (∆I = 33.08% ± 0.28 to 124.91% ± 17.08) and in the resistance to charge transfer (ΔR_CT = 19.73% ± 0.96 to 83.51% ± 0.84). Data analysis revealed high reproducibility, with a relative standard deviation of 3.66%, a response range from 3.58 aM to 357.67 fM, a detection limit of 19.31 aM, and a limit of quantification of 64.39 aM. Therefore, a novel nanosensor for multiparametric electrochemical screening of the TCF3-PBX1 chimeric oncogene was described for the first time, potentially improving the quality of life for leukemic patients. Full article

Background/Objectives: Giardiasis is one of the oldest-reported infectious diseases worldwide. It affects individuals with weakened immune systems and progresses into chronic infection if untreated. Morphological analysis and visualization of cell shapes using unlabeled or fluorophore-labeled samples are commonly employed to identify the parasite. Methods: To distinguish molecular content variations between trophozoites and infectious type I cysts, the current research presents an alternative approach based on label-free Raman microscopy. Results: Constituents responsible for plasma membrane thickening and cyst wall formation during encystation, such as N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) were detected. Although these two coexisting compounds have similar molecular structures, their spectroscopic distinction and visual localization through Raman microscopy are achievable. While immature and non-viable cysts contain a larger amount of GlcNAc, a potential transition of this moiety to GalNAc might occur as the cysts mature and become infectious. Other Raman results revealed changes in the oxidation states of heme-binding proteins and in lipid–protein metabolism, each serving as an additional protection mechanism that the parasite employs for survival. Complementary bright field and confocal fluorescence microscopy results corroborate the Raman outcomes. Conclusions: The molecular-level findings of this work, which presents a detailed spectroscopic analysis of Giardia’s encystation and excystation stages, substantiate the need to use complementary methods for monitoring the parasite’s dynamics and efficacy in terms of self-protection. This alternative method provides accurate insights for further understanding the multifaceted factors involved in Giardia’s encystation process and its acquired resistance to external stimuli. Full article

Sea anemone venoms contain diverse toxins that have significant pharmacological potential, including anticancer, ecticidal, and immunotherapeutic properties. However, critically, the extraction methodology influences venom composition and bioactivity. This study characterized venom from Stichodactyla haddoni obtained via homogenization, electrical stimulation, and milking. Extraction yields varied significantly between methods: the homogenization, electrical stimulation, and milking of healthy sea anemones yielded crude venoms at rates of 17.8%, 3.4%, and 1.5%, respectively. SDS-PAGE revealed distinct protein banding patterns and concentrations, while RP-HPLC demonstrated method-dependent compositional differences. Comprehensive proteomic profiling identified 2370 proteins, encompassing both unique and shared components across extraction techniques. Label-free quantitative analysis confirmed significant variations in protein abundance that was attributable to the extraction method. Cytotoxicity assays against cancer cell lines revealed concentration-dependent inhibition, with milking-derived venom exhibiting the highest potency. Insecticidal activity against Tenebrio molitor was also method-dependent, with milking venom inducing the highest mortality rate. These findings elucidate the profound impact of extraction methodology on the protein composition and functional activities of S. haddoni venom, providing crucial insights for its optimized exploitation in pharmacological development. Full article

The SARS-CoV-2 spike (S1) protein facilitates viral entry through binding to angiotensin-converting enzyme 2 (ACE2), but it also contains an Arg–Gly–Asp (RGD) motif that may enable interactions with RGD-binding integrins on ACE2-negative cells. Here, we provide quantitative evidence for this alternative binding pathway using a live-cell, label-free resonant waveguide grating (RWG) biosensor. RWG technology allowed us to monitor real-time adhesion kinetics of live cells to RGD-displaying substrates, as well as cell adhesion to S1-coated surfaces. To characterize the strength of the integrin–S1 interaction, we determined the dissociation constant using two complementary approaches. First, we performed a live-cell competitive binding assay on RGD-displaying surfaces, where varying concentrations of soluble S1 were added to cell suspensions. Second, we recorded the adhesion kinetics of cells on S1-coated surfaces and fitted the data using a kinetic model based on coupled ordinary differential equations. By comparing the results from both methods, we estimate that approximately 33% of the S1 molecules immobilized on the Nb₂O₅ biosensor surface are capable of initiating integrin-mediated adhesion. These findings support the existence of an alternative integrin-dependent entry route for SARS-CoV-2 and highlight the effectiveness of label-free RWG biosensing for quantitatively probing virus–host interactions under physiologically relevant conditions without the need of the isolation of the interaction partners from the cells. Full article

Full-field optical coherence tomography (FF-OCT) is a high-resolution interferometric imaging technique that enables label-free visualization of cellular structural changes. In this study, we employed a custom-built time-domain FF-OCT system to monitor morphological alterations in HeLa cells undergoing doxorubicin-induced apoptosis and ethanol-induced necrosis at the single-cell level. Apoptotic cells showed characteristic features such as echinoid spine formation, cell contraction, membrane blebbing, and filopodia reorganization. In contrast, necrotic cells exhibited rapid membrane rupture, intracellular content leakage, and abrupt loss of adhesion structure. These dynamic events were visualized using high-resolution tomography and three-dimensional surface topography mapping. Furthermore, FF-OCT-based interference reflection microscopy (IRM)-like imaging effectively highlighted changes in cell–substrate adhesion and cell boundary integrity during the cell death process. Our findings suggest that FF-OCT is a powerful imaging platform for distinguishing cell death pathways and assessing dynamic cellular states, with potential applications in drug toxicity testing, anticancer therapy evaluation, and regenerative medicine. Full article

Accounting for 15–30% of colorectal cancer cases, the serrated pathway remains poorly characterized compared to the adenoma–carcinoma sequence. It involves sessile serrated lesions as precursors and is characterized by BRAF mutations (BRAF^V600E), CpG island hypermethylation, and microsatellite instability (MSI). Using label-free proteomics, we compared normal tissue margins from patients with diverticular disease, sessile serrated lesions, low-grade adenomas, and high-grade adenomas. We identified S100A14 as significantly overexpressed in sessile serrated lesions compared to low-grade adenomas, high-grade adenomas, and normal tissues. This overexpression was confirmed by immunohistochemical scoring in an independent cohort. Gene expression analyses of public datasets showed higher S100A14 expression in BRAF^V600E-mutated and MSI-H colorectal cancers compared to microsatellite stable BRAF^wt tumors. This finding was confirmed by immunohistochemical scoring in an independent colorectal cancer cohort. Furthermore, single-cell RNA sequencing analysis from the Human Colon Cancer Atlas revealed that S100A14 expression in tumor cells positively correlated with the abundance of tumoral CD8⁺ cytotoxic T cells, particularly the CD8⁺ CXCL13⁺ subset, known for its association with a favorable response to immunotherapy. Collectively, our results demonstrate for the first time that S100A14 is a potential biomarker of serrated neoplasia and further suggests its potential role in predicting immunotherapy responses in colorectal cancer. Full article

This study aimed to investigate the underlying mechanisms by which dandelion extract inhibits the proliferation of breast cancer MDA-MB-231 cells. Dandelion root and leaf extracts were prepared using a heat reflux method and subjected to solvent gradient extraction to obtain fractions with different polarities. MTT assays revealed that the ethyl acetate fraction exhibited the strongest inhibitory effect on cell proliferation. LC-MS analysis identified 12 potential active compounds, including sesquiterpenes such as Isoalantolactone and Artemisinin, which showed significantly lower toxicity toward normal mammary epithelial MCF-10A cells compared to tumor cells (p < 0.01). Mechanistic studies demonstrated that the extract induced apoptosis in a dose-dependent manner, with an apoptosis rate as high as 85.04%, and significantly arrested the cell cycle at the S and G2/M phases. Label-free quantitative proteomics identified 137 differentially expressed proteins (|FC| > 2, p < 0.05). GO enrichment analysis indicated that these proteins were mainly involved in cell cycle regulation and apoptosis. KEGG pathway analysis revealed that the antitumor effects were primarily mediated through the regulation of PI3K-Akt (hsa04151), JAK-STAT (hsa04630), and PPAR (hsa03320) signaling pathways. Moreover, differential proteins such as PI3K, AKT1S1, SIRT6, JAK1, SCD, STAT3, CASP8, STAT2, STAT6, and PAK1 showed strong correlation with the core components of the EA-2 fraction of dandelion. Molecular docking results demonstrated that these active compounds exhibited strong binding affinities with key target proteins such as PI3K and JAK1 (binding energy < −5.0 kcal/mol). This study elucidates the multi-target, multi-pathway synergistic mechanisms by which dandelion extract inhibits breast cancer, providing a theoretical basis for the development of novel antitumor agents. Full article

Cancer diagnosis requires alternative techniques that allow for early, non-invasive, or minimally invasive identification. Traditional methods, like tissue biopsies, are highly invasive and can be traumatic for patients. Liquid biopsy, a less invasive option, detects cancer biomarkers in body fluids such as blood and urine. However, early-stage cancer often presents low biomarker levels, making sensitivity a challenge for integrating liquid biopsy into early diagnosis. Recent studies revealed that extracellular vesicles (EVs) secreted by cells are apt markers for liquid biopsy. Detecting extracellular vesicles (EVs) for liquid biopsy faces challenges like low sensitivity, EV subtype heterogeneity, and difficulty isolating pure populations. Label-free methods, such as plasmonic biosensors and Raman spectroscopy, offer potential solutions by enabling direct analysis without markers, improving accuracy, and reducing complexity. This review paper discusses current challenges in EV-based liquid biopsy for cancer diagnosis and prognosis. It addresses the effective use of microfluidics and nano-plasmonic approaches to address these challenges. Enhancing label-free EV detection in liquid biopsy could revolutionize early cancer diagnosis by offering non-invasive, cost-effective, and rapid testing. This could improve patient outcomes through personalized treatment and ease the burden on healthcare systems. Full article

This study aims to investigate the effect of the Pd(II) complex on HeLa cells using computational biology and proteomic analysis. [Pd(dach)Cl₂]-treated HeLa cells were subjected to comparative proteomics analysis using label-free data-independent liquid chromatography-tandem mass spectrometry (LC-MS/MS). In parallel, the informational spectrum method (ISM) was used to predict potential protein interactors of the [Pd(dach)Cl₂] complex in HeLa cells. Proteomics analysis revealed 121 differentially abundant proteins (DAPs). Enrichment analysis of Gene Ontology (GO) annotations revealed ATP hydrolysis and RNA/protein binding as the top molecular functions and RNA splicing and protein–RNA complex organization as the top biological processes. Enrichment analysis of altered canonical pathways pointed out spliceosome and ribosome pathways. The top hub proteins with potential regulatory importance encompassed ribosomal proteins, translational and transcriptional factors, and components of the ribosome assembly machinery. ISM and cross-spectral analysis identified the nucleoplasm and sensor of the single-stranded DNA (SOSS DNA) complex. Proteome analysis showed that [Pd(dach)Cl₂] targets proteins involved in ribosomal biogenesis and RNA splicing, whereas theoretical prediction implies also potential effect on p53 signaling pathway, and thus, alterations of the expression of regulatory proteins involved in cell survival and proliferation. These findings underscore the potential of Pd(II) complexes as anti-cancer agents, warranting further exploration and detailed functional validation. Full article