Search Results (1,584)

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblastic cells in areas of active tissue remodeling, such as wound healing, fibrosis, and certain chronic inflammatory lesions. As FAP is uniquely present in chronic inflammatory lesions and has an important role in extracellular matrix (ECM) turnover, it appears to have all the characteristics necessary for involvement in atherosclerosis and atherosclerotic plaque rupture and has become a potential target in the treatment of myocardial infarction. Methods: To further understand the role of FAP, its expression in atherosclerotic plaques was examined in a genetically modified mouse model of accelerated atherosclerosis (Apobec1 −/− Ldlr −/− double-knockout mice). The immunohistochemical Fap staining of atherosclerotic plaques in a mouse model of atherosclerosis was correlated with quantification of Fap mRNA obtained from the atherosclerotic plaques of the aortic arch. Fap distribution was characterized in mouse atherosclerotic plaques relative to other markers of activated smooth muscle cells, such as alpha smooth muscle actin and myosin heavy chain (Acta2 and Myh2), ECM turnover (Ki-67, procollagen III and Mmp-9), and inflammation in atherosclerosis (Cd-44, Il-12 and Tgf beta) using immunohistochemistry (IH) and RT-PCR analysis. Results: The mouse model of accelerated atherosclerosis showed an increasing presence of Fap with the progression of atherosclerosis and a high expression level in advanced atherosclerotic lesions compared with other markers of ECM turnover and inflammation in atherosclerosis. Conclusions: FAP exhibits a distinct pattern of expression in a mouse model of atherosclerosis as compared to other markers of activated vascular smooth muscle cells, ECM degeneration, and inflammatory cytokines. Full article

Less than 5% of the DNA sequence encodes for proteins, and the remainder encodes for non-coding RNAs (ncRNAs). Among the members of the ncRNA family, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play a pivotal role in the insurgence and progression of several cancers, including leukemia. Thought to have different molecular mechanisms, both miRNAs and lncRNAs act as epigenetic factors modulating gene expression and influencing hematopoietic differentiation, proliferation and immune system function. Here, we discuss the most recent findings on the main molecular mechanisms by which miRNAs and lncRNAs are involved in the pathogenesis and progression of pediatric T acute lymphoblastic leukemia (T-ALL), pointing out their potential utility as therapeutic targets and as biomarkers for early diagnosis, risk stratification and prognosis. miRNAs are involved in the pathogenesis of T-ALL, acting both as tumor suppressors and as oncomiRs. By contrast, to the best of our knowledge, the literature highlights lncRNAs as acting only as oncogenes in this type of cancer by inhibiting apoptosis and promoting cell cycle and drug resistance. Additionally, here, we discuss how these molecules could be detected in the plasma of T-ALL patients, highlighting that lncRNAs may represent a new class of promising accurate and sensitive biomarkers in these young patients. Thus, the unveiling of the aberrant signature of circulating and intracellular levels of lncRNAs could have great clinical utility for obtaining a more accurate definition of prognosis and uncovering novel therapeutic strategies against T-ALL in children. However, further investigations are needed to better define the standard methodological procedure for their quantification and to obtain their specific targeting in T-ALL pediatric patients. Full article

The hydrolysis of raw food sources by commercially available food-grade enzymes releases thousands of peptides. The full characterization of bioactive hydrolysates requires robust methods to identify and quantify key peptides in these food sources. For this purpose, the absolute quantification of specific peptides, part of a complex peptide network, is necessary. Protein quantification with synthetic tryptic peptides as internal standards is a well-known approach, yet the quantification of non-tryptic peptides contained in food hydrolysates is still largely unaddressed. Similarly, data analyses focus on proteomic applications, thus adding challenges to the study of specific peptides of interest. This paper presents an in-sample calibration curve methodology for the identification of three non-tryptic peptides present in a Vicia faba food hydrolysate (PeptiStrong™) using heavy synthetic peptides as both calibrants and internal standards. Full article

Gabapentin (GBP), a γ-aminobutyric acid analogue used for neuropathic pain and epilepsy, can reduce cat stress and improve veterinary exam compliance. A sensitive LC-MS/MS assay was established and rigorously validated for quantifying GBP in feline plasma. The method employed acetonitrile-mediated protein precipitation to efficiently extract GBP and its internal standard, pregabalin (PGB). Chromatographic separation was achieved within 7 min using a C18 column (2.1 × 50 mm, 1.7 μm) with a gradient mobile phase comprising 0.1% (v/v) formic acid in water and acetonitrile. The pharmacokinetics of 25  mg/kg GBP was studied by single-dose oral and intravenous administration. The results demonstrated that the method exhibited satisfactory precision, accuracy and linearity. The pharmacokinetic results showed that the T_max, C_max, T_1/2 and AUC_0–t of GBP in cats after oral administration of 25  mg/kg were (1.83 ± 0.75) h, (13.94 ± 3.75) μg/mL, (5.60 ± 1.79) h and (115.54 ± 27.56) (μg/mL) h, respectively. The results of the study indicated that after intravenous administration of 25 mg/kg GBP, the C₀, T_1/2 and AUC_0–t were (58.82 ± 15.34) μg/mL, (3.87 ± 0.64) h and (160.44 ± 32.65) (μg/mL) h, respectively. The oral bioavailability of GBP in cats was (78.71 ± 18.55)%. In this study, a selective and sensitive LC-MS/MS method for the quantification of GBP in cat plasma was developed and validated. This method was successfully employed to assess the pharmacokinetics following the oral and intravenous administration of GBP in cats. Full article

Forensic biochemistry has often relied on the vitreous humor as a matrix for toxicological investigations due to its stability and isolation from post-mortem redistribution processes. Recently, the scope of research has expanded to explore the vitreous humor as a medium reflecting systemic and pathological changes, particularly in its protein composition. This study delves into the detection and quantification of cardiac damage markers such as CK-MB and myoglobin in vitreous humor samples from 45 autopsy cases. For the first time, it demonstrates a statistically significant correlation between these markers and the perimortem agony interval (PAI), defined as the survival time before death. This discovery paves the way for innovative forensic applications, including the estimation of the PAI, a critical parameter for judicial and compensatory assessments. The findings underscore the potential of the vitreous humor as a diagnostic medium, opening new avenues for understanding the systemic dynamics of cardiac markers and the role of the blood–retinal barrier in post-mortem scenarios. Full article

Glomalin-related soil protein (GRSP), a glycoprotein primarily exuded by arbuscular mycorrhizal fungi (AMF), exerts key roles in ecological processes in terrestrial ecosystems. Nevertheless, the intricate nature of GRSP, coupled with constraints in its extraction and analytical methodologies, impedes a comprehensive understanding of its compositional attributes and ecological functions. Moreover, the scope of current GRSP research has undergone significant expansion, necessitating a comprehensive synthesis in this field. Here, we employed bibliometric analysis to systematically assess research trends and hotspots in the research field of GRSP based on 840 relevant articles indexed in the Web of Science Core Collection database. Among them, key parameters evaluated encompass publications’ quantity, highly cited articles, high-frequency keywords, and historical direct citations. These analyses illuminated the state-of-the-art of GRSP research, delineated emergent trends, and provided future perspectives. Current investigations into GRSP predominantly focus on three major topics: (i) GRSP’s nature, origin, and quantification methodologies; (ii) GRSP’s key influencing factors including agricultural management practices, climate and land use change; and (iii) GRSP’s ecological functions enhancing soil aggregate stability, C sequestration, and contamination remediation. Our findings can serve as a scholarly resource for advancing inquiries into the ecological functionalities of GRSP and its prospective applications in sustainable soil management and ecological restoration. Full article

Background: Every year, over 40 million people sustain mild traumatic brain injury (mTBI) which affects the glucocorticoid stress pathway and synaptic plasticity. Ketamine, a multimodal dissociative anesthetic, modulates the stress pathway and synaptic plasticity. However, the effects of post-mTBI ketamine administration on plasma stress hormones and brain synaptic plasticity are largely unknown. Methods: Adult male Sprague-Dawley rats with indwelling jugular venous catheters sustained mTBI with the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) in a single session (3 impacts × 1.5 J). One hour later, rats received intravenous (IV) ketamine (0, 10, or 20 mg/kg, 2 h). Catheter blood samples were collected for plasma corticosterone and progesterone assays. Brain tissue sections were double-labeled for presynaptic synapsin-1 and postsynaptic density protein 95 (PSD-95). Utilizing the Synaptic Evaluation and Quantification by Imaging Nanostructure (SEQUIN) workflow, super-resolution confocal images were generated, and synapsin-1, PSD-95, and synaptic density were quantified in the CA1 of the hippocampus and medial prefrontal cortex (mPFC). Results: IV ketamine infusion produced biphasic effects on corticosterone levels: a robust elevation during the infusion followed by a reduction after the infusion. CHIMERA injury elevated progesterone levels at post-injury day (PID)-1 and reduced synaptic density in the CA1 at PID-4, regardless of ketamine infusion. Ketamine infusion increased synaptic density in the mPFC at PID-4. Conclusions: Mild TBI and IV ketamine modulate the stress pathway and synaptic plasticity in the brain. Further research is warranted to investigate the functional outcomes of subanesthetic doses of ketamine on stress pathways and neuroplasticity following mTBI. Full article

Introduction: Currently, there are no effective medications for treating all the clinical conditions of patients with COVID-19. We aimed to evaluate the antiviral activity of compounds derived from L-tyrosine against the B.1 lineage of SARS-CoV-2 in vitro and in silico. Methodology: The cytotoxicities of 15 halogenated compounds derived from L-tyrosine were evaluated in Vero-E6 cells by the MTT assay. The antiviral activity of the compounds was evaluated using four strategies, and viral quantification was performed by a plaque assay and qRT-PCR. The toxicity of the compounds was evaluated by ADMET predictor software. The affinity of these compounds for viral or cellular proteins and the stability of their conformations were determined by docking and molecular dynamics, respectively. Results: TODC-3M, TODI-2M, and YODC-3M reduced the viral titer >40% and inhibited the replication of viral RNA without significant cytotoxicity. In silico analyses revealed that these compounds presented low toxicity and binding energies between −4.3 and −5.2 Kcal/mol for three viral proteins (spike, Mpro, and RdRp). TODC-3M and YODC-3M presented the most stable conformations with the evaluated proteins. Conclusions: The most promising compounds were TODC-3M, TODI-2M, and YODC-3M, which presented low in vitro and in silico toxicity, antiviral potential through different strategies, and favorable affinities for viral targets. Therefore, they are candidates for in vivo studies. Full article

Periodontal Ehlers–Danlos syndrome arising from heterozygous pathogenic mutation in C1R and/or C1S genes is an autosomal-dominant disorder characterized by early-onset periodontitis. Due to the difficulties in obtaining and culturing the patient-derived gingival fibroblasts, we established a model system by introducing a heterozygous C1R^R301P/WT mutation into human TERT-immortalized gingival fibroblasts (hGFBs) to investigate its specific effects on collagen metabolism and inflammatory responses. A heterozygous C1R^R301P/WT mutation was introduced into hGFBs using engineered prime editing. The functional consequences of this mutation were assessed at cellular, molecular, and enzymatic levels using a variety of techniques, including cell growth analysis, collagen deposition quantification, immunocytochemistry, enzyme-linked immunosorbent assay, and quantitative real-time reverse transcription polymerase chain reaction. The C1R^R301P/WT-mutated hGFBs (mhGFBs) exhibited normal morphology and growth rate compared to wild-type hGFBs. However, mhGFBs displayed upregulated procollagen α1(V), MMP-1, and IL-6 mRNA expression while simultaneously downregulating collagen deposition and C1r protein levels. A modest accumulation of unfolded collagens was observed in mhGFBs. The mhGFBs exhibited a heightened inflammatory response, with a more pronounced increase in MMP-1 and IL-6 mRNA expression compared to TNF-α/IL-1β-stimulated hGFBs. Unlike cytokine-stimulated hGFBs, cytokine-stimulated mhGFB did not increase C1R, C1S, procollagen α1(III), and procollagen α1(V) mRNA expression. Our results suggest that the C1R^R301P/WT mutation specifically disrupts collagen metabolism and inflammatory pathways in hGFBs, highlighting the mutation’s role in these processes. While other cellular functions appear largely unaffected, these findings underscore the potential of targeting collagen metabolism and inflammation for therapeutic interventions in pEDS. Full article

Background: Phytochemicals possess diverse therapeutic potential; however, the impact of arene substitutions on the pharmacological properties of the bibenzyl compounds batatasin III and gigantol, derived from Dendrobium venustum, remains unexplored. Objectives: This study examines how structural differences between these compounds affect cellular glucose uptake and lipid metabolism during adipocyte differentiation. Methods: The effects of both bibenzyl compounds on cytotoxicity and glucose uptake were assessed in mouse and human pre-adipocytes and rat skeletal muscle myoblasts using colorimetric assays. Lipid metabolism was evaluated through Oil Red O staining and quantification of triglyceride and glycerol levels, while protein and gene expression during adipocyte differentiation were analyzed via western blotting and RT-qPCR. Results: At the highest non-cytotoxic concentration (25 µM), gigantol significantly enhanced glucose uptake (up to 2-fold) under both basal and insulin-stimulated conditions, whereas batatasin III showed a similar effect only under basal conditions. Gigantol upregulated GLUT1 and GLUT4 in myotubes but downregulated them in adipocytes, whereas batatasin III had minimal impact on these transporters. Both compounds suppressed lipid accumulation in mouse and human adipocytes by decreasing intracellular triglyceride content and promoting extracellular glycerol release. However, batatasin III did not affect extracellular glycerol release during early adipocyte differentiation, as evidenced by the marked downregulation of key lipogenic proteins (PLIN1, LPL, FABP4) observed only with gigantol. Molecular docking analyses suggest that gigantol’s greater bioactivity may result from its higher number of arene substitutions. Conclusions: This study provides the first evidence that differences in arene substitutions among orchid-derived bibenzyls influence their pharmacological properties. Our findings support the strategic modification of natural products as a potential approach for managing metabolic disorders. Full article

Red meat consumption is considered a risk factor for colorectal cancer (CRC) development and stimulated isolation of plasmid-like DNA molecules from bovine serum and milk, termed bovine meat and milk factors (BMMFs). BMMFs encode a conserved replication protein (Rep). Increased populations of Rep-expressing macrophages have been identified in the peritumor of CRC patients and pre-cancerous tissues when compared to the tissues of healthy individuals. This supports the concept that BMMFs increase cancer risk by indirect carcinogenesis, upon induction of chronic inflammation. However, the spread of Rep⁺ immune cells in tissues at greater distances from primary tumors has not yet been assessed. Here, we immunohistologically analyzed the presence of Rep⁺ immune cells in sets of tumor, peritumor and, additionally, distant tissues of CRC patients (n = 13). We identified consistently high numbers of BMMF-positive macrophages in mucosal tissues at distances of as much as 25 cm away from the primary tumors, at levels comparable to peritumors and associated with M2-like macrophage polarization. The broad distribution of BMMFs suggests that BMMF⁺ macrophages might already exist at stages of pre-cancerous dysplasia or before. Quantification of BMMF tissue expression during colonoscopy might help to preventively stratify individuals at risk of developing polyps/CRC and recommend them for enhanced surveillance and/or changes in dietary lifestyle. Full article

Plants are challenged regularly with multiple types of biotic stress factors, such as pathogens or insect herbivores, in their environment. To detect and defend against pathogens, plants have evolved an innate immune system in which intracellular receptors in the so-called effector-triggered immunity play a vital role. In Arabidopsis thaliana the Toll/interleukin-1 receptors (TIRs) domain is related to intracellular immunity receptors, for example in TIR-NBS-LRR (TNL) proteins. Among the TIR domain carrying proteins, very little is known about the function of the TIR-X proteins. Here, we focus on the recently described TIR-X (TIRP; At5g44900) to analyze its role in phytohormone-mediated plant defense through gene expression and phytohormone quantification. Therefore, we employed two fungal pathogens, the necrotrophic Alternaria brassicicola and the hemibiotrophic Verticillium dahliae, to infect A. thaliana WT (Col-0), TIRP knock-out, and TIRP overexpressing lines for comparative analyses. Furthermore, we included the insect herbivore Spodoptera littoralis and a treatment with S. littoralis egg extract on the plants to analyze any role of TIRP during these attacks. We found that both A. brassicicola and V. dahliae infections increased TIRP gene expression systemically. The salicylic acid content was higher in the TIRP overexpressing line, corresponding to a better S. littoralis larval growth performance in feeding assays. However, since we never observed clear infection-related differences in jasmonate or salicylic acid levels between the wild type and the two transgenic Arabidopsis lines, our results rule out the possibility that TIRP acts via the regulation of phytohormone synthesis and accumulation. Full article

Somatic embryogenesis (SE) involves the formation of embryo-like structures from somatic cells without fertilization and is widely used for clonal propagation and genetic transformation. However, in olive (Olea europaea sp. europaea), SE remains challenging due to the recalcitrant behavior of adult tissues when used as initial explants. Bioactive molecules released into the culture medium (conditioned medium, CM) by embryogenic cultures have been identified as modulators of the SE response. However, their potential role in enhancing SE efficiency in olive and overcoming tissue recalcitrance remains largely unexplored. To investigate the role of these biomolecules in olive SE, a protocol was established using SE cultures of cv. ‘Galega Vulgar’. Proteins and metabolites were separated by filtration, concentrated through lyophilization, and precipitated using three methods: Acetone, TCA/Acetone, and Methanol/Chloroform. The efficiency of these methods was evaluated through total protein quantification and via SDS-PAGE electrophoresis. LC-MS/MS was employed to analyze secretome composition using the TCA/Acetone precipitation method. Additionally, metabolite profiles were analyzed using ¹H NMR spectroscopy. The results led to the identification of 1096 (526 protein groups) Olea europaea proteins, including well-known SE biomarkers such as kinases and peroxidases. NMR spectroscopy identified several metabolites secreted into the medium or resulting from the metabolic activity of secreted enzymes, confirming the applicability of the procedure. Although extracting secreted biomolecules from the culture medium presents significant challenges, the protocol established in this study successfully enabled the isolation and identification of both proteins and metabolites, revealing a valuable workflow for future in-depth analyses of secreted biomolecules in olive SE. Full article

An Mg-based alloy device manufactured via a superplastic forming process (Mg-AZ31+SPF) and coated using a hydrothermal method (Mg AZ31+SPF+HT) was investigated as a method to increase mechanical and osteointegration capability. The cell viability and osteointegrative properties of alloy-derived Mg AZ31+SPF and Mg AZ31+SPF+HT extracts were investigated regarding their effect on human mesenchymal stem cells (hMSCs) (maintained in basal (BM) and osteogenic medium (OM)) after 7 and 14 days of treatment. The viability was analyzed through metabolic activity and double-strand DNA quantification, while the osteoinductive effects were evaluated through qRT-PCR, osteoimage, and BioPlex investigations. Finally, a preliminary liquid mass spectrometry analysis was conducted on the secretome of hMSCs. Biocompatibility analysis revealed no toxic effect on cells’ viability or proliferation during the experimental period. A modulation effect was observed on the osteoblast pre-commitment genes of hMSCs treated with Mg-AZ31+SPF+HT in OM, which was supported by mineralization nodule analysis. A preliminary mass spectrometry investigation highlighted the modulation of protein clusters involved in extracellular exosomes, Hippo, and the lipid metabolism process. In conclusion, our results revealed that the Mg AZ31+SPF+HT extracts can modulate the canonical and non-canonical osteogenic process in vitro, suggesting their possible application in bone tissue engineering. Full article

The accurate quantification of food allergens is crucial for ensuring consumer safety and compliance with regulatory requirements. A proficiency test (PT) was organised to evaluate the performance of laboratories in quantifying total egg and total milk protein in cookies. The PT involved 20 laboratories, which reported results using mainly commercial ELISA kits and liquid chromatography–tandem mass spectrometry (LC-MS/MS). The findings indicate a satisfactory performance for milk protein determination among the majority of participant laboratories. However, the quantification of egg proteins in heated products remains a challenge, with most laboratories reporting results significantly below the reference value. Several potential factors contributing to this challenge are discussed, including the denaturation of egg proteins during heat treatment, differences in extraction strategies and the antibodies used in ELISA kits, and the lack of standardised methods and conversion factors for LC-MS/MS analysis. These findings underscore the importance of regular PT exercises to evaluate laboratory performance and ensure compliance with WHO/FAO recommendations. The results of this study aim to guide the development of improved analytical methods and strategies for ensuring the accurate quantification of food allergens. Full article