Search Results (4)

Severe traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide. To date, there are no clinically effective neuroprotective agents. Biomaterials that combine structural support for damaged tissue with a depot for therapeutic agents may represent a key solution to this problem. To evaluate the neuroprotective potential of a collagen matrix derived from the jellyfish Rhizostoma pulmo (R. pulmo) and modified with sodium thiosulfate (Na₂S₂O₃) as an hydrogen sulfide (H₂S) donor in a bioengineered platform for the treatment of severe TBI. Comprehensive characterization of the collagen matrix (electrophoresis, fluorescence microscopy), its implantation in a mouse model of severe TBI, and subsequent morphological, histological, ultrastructural, and immunohistochemical analyses of connexin 43 (Cx43) and p53 protein (p53) were performed. In addition, molecular dynamics simulations of the interactions between sulfur-containing compounds and target proteins were conducted. The effects were compared with inhibition of endogenous H₂S synthesis using aminooxyacetic acid (AOAA). The collagen matrix retains the properties of type I collagen and forms a three-dimensional porous structure with high hydrophilicity and biocompatibility. Implantation ensures effective defect filling, reduces cystic degeneration, and preserves cortical structure. Modification with Na₂S₂O₃ results in a significant reduction in both nuclear and cytoplasmic accumulation of p53, prevention of Cx43 dysregulation, a decrease in the proportion of damaged neurons and inflammatory infiltration, and preservation of tissue ultrastructure. In contrast, inhibition of CBS with AOAA exacerbates pathological changes. Molecular modeling demonstrated that S₂O₃²⁻ is capable of forming stable electrostatic interactions with domains of p53 and Cx43 under conditions of acidosis and elevated Ca²⁺. A collagen matrix derived from R. pulmo and modified with Na₂S₂O₃ represents a promising biodegradable platform that combines structural support with local H₂S-dependent regulation of key mechanisms of secondary brain injury. This approach provides a multilevel neuroprotective effect and opens new opportunities for the development of therapeutic implants for severe TBI. Full article

Affinity gel electrophoresis was introduced about 50 years ago. Proteins interact with a ligand immobilized in the support. Specific interactions cause a decrease in electrophoretic mobility. The presence of a free ligand, competing with an immobilized ligand, restores electrophoretic mobility. In affinity capillary electrophoresis, the ligand is mobile, and its interaction with a specific protein changes the mobility of the protein–ligand complex. This review mostly focuses on gel affinity electrophoresis. The theoretical basis of this technique, ligand immobilization strategies, and principles for determination of ligand affinity are addressed. Factors affecting specificity and strength of interactions are discussed, in particular, the structure of the affinity matrix, pH, temperature, hydrostatic pressure, solvent, co-solvents, electric field, and other physico-chemical conditions. Capillary affinity electrophoresis principles and uses are also briefly introduced. Affinity gel electrophoresis can be used for qualitative and quantitative purposes. This includes detection of specific proteins in complex media, investigation of specific interactions, protein heterogeneity, molecular and genetic polymorphism, estimation of dissociation constants of protein–ligand complexes, and conformational stability of binding sites. Future prospects, in particular for screening of engineered mutants and potential new drugs, coupling to other analytical methods, and ultra-microtechnological developments, are addressed in light of trends and renewal of this old technique. Full article

Fasciola hepatica is the main causative agent of fasciolosis, a zoonotic parasitic disease of growing public health concern. F. hepatica metacercariae are ingested by the host and excyst in the intestine, thereby releasing the newly excysted juveniles (FhNEJ), which traverse the gut wall and migrate towards the biliary ducts. Since blocking F. hepatica development is challenging after crossing of the intestinal wall, targeting this first step of migration might result in increased therapeutic success. The intestinal extracellular matrix (ECM) is constituted by a network of structural proteins, including laminin (LM) and fibronectin (FN), that provide mechanical support while acting as physical barrier against intestinal pathogens. Here, we employed ELISA and immunofluorescent assays to test for the presence of LM- and FN-binding proteins on a tegument-enriched antigenic fraction of FhNEJ, and further determined their identity by two-dimensional electrophoresis coupled to mass spectrometry. Additionally, we performed enzymatic assays that revealed for the first time the capability of the juvenile-specific cathepsin L3 to degrade LM, and that LM degradation by FhNEJ proteins is further potentiated in the presence of host plasminogen. Finally, a proteomic analysis showed that the interaction with LM triggers protein changes in FhNEJ that may be relevant for parasite growth and adaptation inside the mammalian host. Altogether, our study provides valuable insights into the molecular interplay between FhNEJ and the intestinal ECM, which may lead to the identification of targetable candidates for the development of more effective control strategies against fasciolosis. Full article

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. Full article