Biomolecules最新文献

The microbiota-gut-brain axis is a complex bidirectional communication system that involves multiple interactions between intestinal functions and the emotional and cognitive centers of the brain. These interactions are mediated by molecules (metabolites) produced in both areas, which are considered mediators. To shed light on this complex mechanism, which is still largely unknown, a reliable characterization of the mediators is essential. Here, we review the most studied metabolites in the microbiota-gut-brain axis, the metabolic pathways in which they are involved, and their functions. This review focuses mainly on the use of mass spectrometry for their determination, reporting on the latest analytical methods, their limitations, and future perspectives. The analytical strategy for the qualitative-quantitative characterization of mediators must be reliable in order to elucidate the molecular mechanisms underlying the influence of the above-mentioned axis on stress resilience or vulnerability.

Nitrate (NO₃) and nitrite (NO₂) are important nitrogen compounds that play a vital role in the nitrogen cycle, contributing to plant nutrition and broader ecological functions. Nitrates are produced from nitric acid (HNO₃), while nitrites come from nitrous acid (HNO₂). These substances are commonly found in the environment, especially in food and water, due to contamination from both human and natural sources. Human activities are major contributors to the high levels of nitrates found in water, leading to environmental pollution. Although nitrogen is crucial for plant growth, excessive fertilizer use has caused ecological disruptions. In plants, nitrates tend to accumulate primarily in the leaves of non-leguminous crops, such as leafy vegetables, which are known for their high nitrate content. Furthermore, nitrates and nitrites are added to animal-based foods, especially processed meats and cheeses, to prevent bacterial growth, slow spoilage, and improve flavor and color. The concentration of these compounds in food can vary due to different factors like farming practices, climate, soil conditions, and food production methods. This review seeks to examine the differences between the plant-based and animal-based sources of these compounds and assess their potential impact on human health, considering also the paradigm that goes beyond nitric oxide production.

The family of the β₂-integrin receptors is critically involved in host defense and homeostasis, by mediating immune cell adhesion, migration, and phagocytosis. Due to their key roles in immune surveillance and inflammation, their modulation has been recognized as an attractive drug target. However, the development of therapeutics has been limited, partly due to the high promiscuity of endogenous ligands, their functional responses, and gaps in our understanding of their disease-related molecular mechanisms. The delineation of the molecular role of β₂ integrins and their ligands has been hampered by a shortage of validated assay systems. To facilitate molecular and functional studies on the β₂-integrin family, and to enable screening of modulators, this study provides a uniform and validated assay platform. For this purpose, the major ligand-binding domains (αI) of all four β₂ integrins were recombinantly expressed in both low- and high-affinity states. By optimizing the expression parameters and selecting appropriate purification tags, all αI-domain variants could be produced with high yield and purity. Direct binding studies using surface plasmon resonance (SPR) confirmed the expected activity and selectivity profiles of the recombinant αI domains towards their reported ligands, validating our approach. In addition, the SPR studies provided additional insights into ligand binding, especially for the scarcely described family member CD11d. Alongside characterizing endogenous ligands, the platform can be employed to test pharmacologically active compounds, such as the reported β₂-integrin antagonist simvastatin. In addition, we established a bead-based adhesion assay using the recombinant αI domains, and a cell-based adhesion assay underlining most findings generated with the isolated αI domains. Interestingly, the binding of ligands to the recombinant α_DI is not dependent on divalent cation, in contrast to the full integrin CD11d/CD18, suggesting a binding mode distinct of the metal ion-dependent adhesion site (MIDAS). The setup highlights the applicability of recombinant αI domains for first screenings and direct or competitive interaction studies, while the full integrin is needed to validate those findings.

Cancer management has traditionally depended on chemotherapy as the mainstay of treatment; however, recent advancements in targeted therapies and immunotherapies have offered new options. Ubiquitin-specific proteases (USPs) have emerged as promising therapeutic targets in cancer treatment due to their crucial roles in regulating protein homeostasis and various essential cellular processes. This review covers the following: (1) the structural and functional characteristics of USPs, highlighting their involvement in key cancer-related pathways, and (2) the discovery, chemical structures, mechanisms of action, and potential clinical implications of USP inhibitors in cancer therapy. Particular attention is given to the role of USP inhibitors in enhancing cancer immunotherapy, e.g., modulation of the tumor microenvironment, effect on regulatory T cell function, and influence on immune checkpoint pathways. Furthermore, this review summarizes the current progress and challenges of clinical trials involving USP inhibitors as cancer therapy. We also discuss the complexities of achieving target selectivity, the ongoing efforts to develop more specific and potent USP inhibitors, and the potential of USP inhibitors to overcome drug resistance and synergize with existing cancer treatments. We finally provide a perspective on future directions in targeting USPs, including the potential for personalized medicine based on specific gene mutations, underscoring their significant potential for enhancing cancer treatment. By elucidating their mechanisms of action, clinical progress, and potential future applications, we hope that this review could serve as a useful resource for both basic scientists and clinicians in the field of cancer therapeutics.

The p21-activated kinase (PAK1), a serine/threonine protein kinase, is critical in regulating various cellular processes, including muscle contraction, neutrophil chemotaxis, neuronal polarization, and endothelial barrier function. Aberrant PAK1 activity has been implicated in the progression of several human diseases, including cancer, heart disease, and neurological disorders. Increased PAK1 expression is often associated with poor clinical prognosis, invasive tumor characteristics, and therapeutic resistance. Despite its importance, the cellular mechanisms that modulate PAK1 function remain poorly understood. Accessory proteins, essential for the precise assembly and temporal regulation of signaling pathways, offer unique advantages as therapeutic targets. Unlike core signaling components, these modulators can attenuate aberrant signaling without completely abolishing it, potentially restoring signaling to physiological levels. This review highlights PAK1 accessory proteins as promising and novel therapeutic targets, opening new horizons for disease treatment.

Metformin is the first-line medication for treating type 2 diabetes mellitus, with more than 200 million patients taking it daily. Its effects are extensive and play a positive role in multiple areas. Can its effects and potential mechanisms be explored through the urine proteome? In this study, 166 differential proteins were identified following the administration of 150 mg/(kg·d) of metformin to rats for five consecutive days. These included complement component C6, pyruvate kinase, coagulation factor X, growth differentiation factor 15, carboxypeptidase A4, chymotrypsin-like elastase family member 1, and L-lactate dehydrogenase C chain. Several of these proteins have been reported to be directly affected by metformin or associated with its effects. Multiple biological pathways enriched by these differential proteins, or proteins containing differentially modified peptides, have been reported to be associated with metformin, such as the glutathione metabolic process, negative regulation of gluconeogenesis, and the renin-angiotensin system. Additionally, some significantly changed proteins and enriched biological pathways, not yet reported to be associated with metformin's effects, may provide clues for exploring its potential mechanisms. In conclusion, the application of the urine proteome offers a comprehensive and systematic approach to exploring the effects of drugs, providing a new perspective on the study of metformin's mechanisms.

Mastitis is the leading cause of economic losses in dairy farming, significantly impairing animal welfare and the quality and quantity of milk production. MicroRNAs are increasingly gaining attention, in both human and veterinary medicine, as biomarkers for various diseases. This study evaluated the diagnostic potential of four circulating microRNAs (miR-26-5p, miR-142-5p, miR-146a, and miR-223-3p) by examining changes in their expression in milk samples from dairy cows at different immune-cell subpopulations correlated to different stage of mastitis with a validated method. Additionally, this study has analyzed the possible source of these circulating microRNAs by the measurement of their secretion from activated immune cells (lymphocytes, monocytes, and neutrophils). miR-223-3p has been significantly expressed in an acute stage of mastitis (p < 0.01) but not in the chronic or susceptible stages. Conversely, mir-26-5p has been significantly reduced in acute, chronic, and susceptible groups of animals. In immune-cell cultures, miR-26 has been shown to be down-regulated in lipopolysaccharide (LPS)-stimulated neutrophils, while miR-223 has been shown to be up-regulated in phytohemagglutinin (PHA)-stimulated lymphocytes. The differential expression of miR-223-3p and miR-26-5p, combined with differential and total somatic cell count, could serve as a useful tool for identifying the evolutionary stage of mastitis-related inflammatory pathology.

Cardiovascular diseases, including atherosclerosis, hypertension, and heart failure, remain the leading cause of global mortality, with endothelial dysfunction and vascular remodeling as critical contributors. Integrins, as transmembrane adhesion proteins, are central regulators of cell adhesion, migration, and signaling, playing a pivotal role in maintaining vascular homeostasis and mediating pathological processes such as inflammation, angiogenesis, and extracellular matrix remodeling. This article comprehensively examines the role of integrins in the pathogenesis of cardiovascular diseases, focusing on their dysfunction in endothelial cells and interactions with inflammatory mediators, such as TNF-α. Molecular mechanisms of integrin action are discussed, including their involvement in mechanotransduction, leukocyte adhesion, and signaling pathways that regulate vascular integrity. The review also highlights experimental findings, such as the use of specific integrin-targeting plasmids and immunofluorescence to elucidate integrin functions under inflammatory conditions. Additionally, potential therapeutic strategies are explored, including the development of integrin inhibitors, monoclonal antibodies, and their application in regenerative medicine. These approaches aim not only to mitigate pathological vascular remodeling but also to promote tissue repair and angiogenesis. By bridging insights from molecular studies with their translational potential, this work underscores the promise of integrin-based therapies in advancing the management and treatment of cardiovascular diseases.

Circular RNAs (circRNAs) have attracted increasing attention for their roles in human diseases, making the prediction of circRNA-disease associations (CDAs) a critical research area for advancing disease diagnosis and treatment. However, traditional experimental methods for exploring CDAs are time-consuming and resource-intensive, while existing computational models often struggle with the sparsity of CDA data and fail to uncover potential associations effectively. To address these challenges, we propose a novel CDA prediction method named the Graph Isomorphism Transformer with Dual-Stream Neural Predictor (GIT-DSP), which leverages knowledge graph technology to address data sparsity and predict CDAs more effectively. Specifically, the model incorporates multiple associations between circRNAs, diseases, and other non-coding RNAs (e.g., lncRNAs, and miRNAs) to construct a multi-source heterogeneous knowledge graph, thereby expanding the scope of CDA exploration. Subsequently, a Graph Isomorphism Transformer model is proposed to fully exploit both local and global association information within the knowledge graph, enabling deeper insights into potential CDAs. Furthermore, a Dual-Stream Neural Predictor is introduced to accurately predict complex circRNA-disease associations in the knowledge graph by integrating dual-stream predictive features. Experimental results demonstrate that GIT-DSP outperforms existing state-of-the-art models, offering valuable insights for precision medicine and disease-related research.

Cry j 7 is a 7 kDa cysteine-rich gibberellin regulatory protein (GRP) with six disulfide bonds. It was isolated from Japanese cedar as the pollen allergen in this study. It exhibits cross-reactivity with food allergens such as Pru p 7 from peach and causes pollen-food allergy syndrome (PFAS). In this work, recombinant Cry j 7 and Pru p 7 were successfully overexpressed using Pichia pastoris in a high-cell-density fermentation culture, and pure proteins were purified by reverse-phase HPLC. The characterization of Cry j 7 and Pru p 7 were performed by MS, CD, and ¹H-NMR experiments to confirm the correct native conformation of Cry j 7 as well as Pru p 7. When compared, the results showed that Cry j 7 exhibits excellent stability in disulfide linkages and preserves its original structure up to 90 °C in various pH buffers in comparison to Pru p 7. Notably, NMR analyses indicated the greater mobility in the α-helix and loop regions of S38-C47 in Pru p 7 compared to those of Cry j 7. Furthermore, our results showed that the sensitivity of Cry j 7 to enzyme digestion differed from that of Pru p 7: Cry j 7 was more susceptible to proteolysis, while Pru p 7 displayed better resistance in the gastrointestinal tract. These variations in structural stability and sensitivity to proteolysis provide valuable insights into the allergenicity within the GRP family.