Search Results (36)

Antibiotic stereoisomers as components of medicines are typically characterized by different biological activities. Because pharmaceuticals can include a racemic mixture of stereoisomers, monitoring of all forms is required. One contaminant of food products, antibiotic ofloxacin (OFL), as a chiral compound, has two enantiomers—the biologically active S-isomer and less active R-isomer. In this study, a sensitive immunochromatographic test system for simultaneous enantiospeсific detection of the two OFL isomers was developed for the first time. For this, polyclonal antibodies were produced, and conditions for a double lateral flow immunoassay (LFIA) were selected and optimized so that the cross-reactivity with another enantiomer was negligible. The LFIA was performed in a competitive format with gold nanoparticles as a label for secondary antibodies. The achieved LODs/cutoffs were 0.001/10 and 0.007/30 ng/mL for S-OFL and R-OFL detection, respectively; the assay procedure took only 15 min. A double LFIA was performed to detect S-OFL and R-OFL in milk with minimal sample pretreatment; the recoveries were 85–95%. The developed test system is an effective tool for the selective detection of both isomers of OFL, allowing for the avoidance of false negative results. This immunochromatographic approach can be promising for the control of other optically active food toxicants. Full article

Sugarcane streak mosaic virus (SCSMV) is one of the mosaic viruses found in mixed infection with two or more viruses. Infections of mosaic viruses show highly similar mosaic symptoms and are difficult to distinguish. This study aimed to develop a specific antibody against SCSMV that could potentially be utilized to differentiate mosaic virus infections. The cDNA encoding the coat protein (CP) of SCSMV was isolated by RT-PCR from the symptomatic sugarcane leaves. The cDNA was then used for the production of CP for the development of its polyclonal antibody. The nucleotide sequence of the cDNA showed high homology of 94.2–97.3% at the amino acid level with the CP-cDNA of SCSMV isolated from India (AM749403.1), Africa (OR195142.1), and USA (U75456.1). CP was produced as a recombinant protein with a molecular size of 36.5 kDa in Escherichia coli. The injection of recombinant CP into a rabbit resulted in the production of polyclonal antibodies, which were used for the immunodetection of SCSMV in sugarcane. Immunoblot analysis revealed a specific reaction of SCSMV-CP in symptomatic sugarcane leaves. ELISA (Enzyme-Linked Immunosorbent Assay) and IC-RT-PCR (Immunocapture-Reverse Transcription-Polymerase Chain Reaction) using the CP antibody proved successful for detecting SCSMV infection in sugarcane leaves. The results indicate that the SCSMV-CP antibody is suitable for an immunodetection system and exhibits high specificity for SCSMV infection. Full article

Amaranth is a promising staple food that produces seeds with excellent nutritional quality. Although cultivated species intended for grain production have interesting agronomic traits, relatively little is known about wild species, which can prosper in diverse environments and could be a rich genetic source for crop improvement. This work focuses on the proteomic comparison between the seeds of wild and cultivated amaranth species using polarity-based protein extraction and two-dimensional gel electrophoresis. Differentially accumulated proteins (DAPs) showed changes in granule-bound starch synthases and a wide range of 11S globulin isoforms. The electrophoretic profile of these proteins suggests that they may contain significant phosphorylation as post-translational modifications (PTMs), which were confirmed via immunodetection. These PTMs may impact the physicochemical functionality of storage proteins, with potential implications for seed agronomic traits and food system applications. Low-abundant DAPs with highly variable accumulation patterns are also discussed; these were involved in diverse molecular processes, such as genic regulation, lipid storage, and stress response. Full article

Tissue homeostasis, function recovery, and protection mechanisms are boosted by the balanced and timely control of inflammation and oxidative stress. Nowadays, many natural products and bio-derivates exhibit antioxidant and anti-inflammatory activity, supporting medical care and tissue wellness against inflammation, oxidative stress, and inflammaging. Castanea sativa wood distillate (WD) is a bio-derivative used as a corroborant and biofertilizer in agriculture. Based on the safety profile of low concentrations of WD on human cells, the present study aims to assess the anti-inflammatory and antioxidant activity of WD on different cell types in the integumentary system. Human keratinocytes, mucosal epithelium, dermal fibroblasts, and endothelial cells were exposed to WD, and the concentrations devoid of pro-apoptotic potential were profiled. Then, the effect of nontoxic doses of WD revealed an anti-inflammatory effect, observed through the immunodetection of prostanoid cascade markers in experimentally induced inflammation. A reduction in endothelial hyperpermeability was evidenced by the immunofluorescence analysis of cell–cell adhesion proteins, VE-cadherin and ZO-1. In addition, WD buffered the exogenously produced oxidative stress. On the whole, WD showed both anti-inflammatory and antioxidant activities on the various cell types, preserving endothelial barrier integrity. Overall, this study supports the involvement of this bio-derivative in novel exploitable fields, such as therapeutic dermatological applications for human and animal medical care. Full article

Anderson-Fabry disease (AFD), a genetic disorder caused by mutations in the α-galactosidase-A (GLA) gene, disrupts lysosomal function, leading to vascular complications. The accumulation of globotriaosylceramide (Gb3) in arterial walls triggers upregulation of adhesion molecules, decreases endothelial nitric oxide synthesis, and induces reactive oxygen species production. This cascade results in fibrotic thickening, endothelial dysfunction, hypercontractility, vasospasm, and a pro-thrombotic phenotype. AFD patients display increased intima-media thickness (IMT) and reduced flow-mediated dilation (FMD), indicating heightened cardiovascular risk. Nailfold capillaroscopy (NFC) shows promise in diagnosing and monitoring microcirculatory disorders in AFD, though it remains underexplored. Morphological evidence of AFD as a storage disorder can be demonstrated through electron microscopy and immunodetection of Gb3. Secondary pathophysiological disturbances at cellular, tissue, and organ levels contribute to the clinical manifestations, with prominent lysosomal inclusions observed in vascular, cardiac, renal, and neuronal cells. Chronic accumulation of Gb3 represents a state of ongoing toxicity, leading to increased cell turnover, particularly in vascular endothelial cells. AFD-related vascular pathology includes increased renin-angiotensin system activation, endothelial dysfunction, and smooth muscle cell proliferation, resulting in IMT increase. Furthermore, microvascular alterations, such as atypical capillaries observed through NFC, suggest early microvascular involvement. This review aims to unravel the complex interplay between inflammation, oxidative stress, and endothelial dysfunction in AFD, highlighting the potential connections between metabolic disturbances, oxidative stress, inflammation, and fibrosis in vascular and cardiac complications. By exploring novel cardiovascular risk factors and potential diagnostic tools, we can advance our understanding of these mechanisms, which extend beyond sphingolipid accumulation to include other significant contributors to disease pathogenesis. This comprehensive approach can pave the way for innovative therapeutic strategies and improved patient outcomes. Full article

Organic electrochemical transistors appear as an alternative for relatively low-cost, easy-to-operate biosensors due to their intrinsic amplification. Herein, we present the fabrication, characterization, and validation of an immuno-detection system based on commercial sensors using gold electrodes where no additional surface treatment is performed on the gate electrode. The steady-state response of these sensors has been studied by analyzing different semiconductor organic channels in order to optimize the biomolecular detection process and its the application to monitoring human IgG levels due to SARS-CoV-2 infections. Detection levels of up to tens of $\mathsf{\mu }\mathrm{g}{\mathrm{mL}}^{-1}$ with sensitivities up to 13.75% [$\mathsf{\mu }\mathrm{g}/\mathrm{mL}$]⁻¹, concentration ranges of medical relevance in seroprevalence studies, have been achieved. Full article

This study was aimed at the sensitive immunodetection of porcine myoglobin (MG) as a species-specific biomarker in meat products. The enhanced lateral flow immunoassay (LFIA) was created in the sandwich format using monoclonal antibodies (Mab) with specificity to porcine MG and labeled by Prussian blue nanoparticles (PBNPs) as peroxidase-mimicking nanozymes. Signal amplification was provided by the colored product of oxidation catalyzed by the PBNPs. Several Mab–PBNP conjugates with different antibody loads were synthesized; the one that provided the best analytical characteristics of the LFIA was selected. Advanced optimization of the test system was carried out. As a result, the visual limit of detection (LOD) of MG was 1.5 ng/mL. Involvement of the catalytic nanozyme properties allowed the LOD to be decreased by ~9 times in comparison to the LFIA based on gold nanomarkers, and by ~27 times compared to the LFIA based on PBNP coloration. The assay time was 30 min, including catalytic enhancement. A simple technique of meat sample pre-treatment aimed at effective MG extraction and matrix disposal was proposed. The specificity of the LFIA towards the pork meat was demonstrated. The applicability of the created test system was shown by testing extracts obtained from finished meat products. Full article

Epigenetic alterations contribute to the pathogenesis of chronic diseases such as diabetes mellitus. Previous studies of our group showed that diabetic conditions reduce the trimethylation of H3K27 in podocytes in a NIPP1- (nuclear inhibitor of protein phosphatase 1) and EZH2- (enhancer of zeste homolog 2) dependent manner. It has been previously reported that in differentiated podocytes, hypoxia decreases the expression of slit diaphragm proteins and promotes foot process effacement, thereby contributing to the progression of renal disease. The exact mechanisms are, however, not completely understood. The aim of this study was to analyze the role of hypoxia and HIFs (hypoxia-inducible factor) on epigenetic changes in podocytes affecting NIPP1, EZH2 and H3K27me3, in vitro and in vivo. In vivo studies were performed with mice exposed to 10% systemic hypoxia for 3 days or injected with 3,4-DHB (dihydroxybenzoate), a PHD (prolyl hydroxylase) inhibitor, 24 h prior analyses. Immunodetection of H3K27me3, NIPP1 and EZH2 in glomerular podocytes revealed, to the best of our knowledge for the first time, that hypoxic conditions and pharmacological HIFs activation significantly reduce the expression of NIPP1 and EZH2 and diminish H3K27 trimethylation. These findings are also supported by in vitro studies using murine-differentiated podocytes. Full article

Parkinson’s Disease (PD), treated with the dopamine precursor l-3,4-dihydroxyphenylalanine (L-DOPA), displays motor and non-motor orofacial manifestations. We investigated the pathophysiologic mechanisms of the lateral pterygoid muscles (LPMs) and the trigeminal system related to PD-induced orofacial manifestations. A PD rat model was produced by unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. Abnormal involuntary movements (dyskinesia) and nociceptive responses were determined. We analyzed the immunodetection of Fos-B and microglia/astrocytes in trigeminal and facial nuclei and morphological markers in the LPMs. Hyperalgesia response was increased in hemiparkinsonian and dyskinetic rats. Hemiparkinsonism increased slow skeletal myosin fibers in the LPMs, while in the dyskinetic ones, these fibers decreased in the contralateral side of the lesion. Bilateral increased glycolytic metabolism and an inflammatory muscle profile were detected in dyskinetic rats. There was increased Fos-B expression in the spinal nucleus of lesioned rats and in the motor and facial nucleus in L-DOPA-induced dyskinetic rats in the contralateral side of the lesion. Glial cells were increased in the facial nucleus on the contralateral side of the lesion. Overall, spinal trigeminal nucleus activation may be associated with orofacial sensorial impairment in Parkinsonian rats, while a fatigue profile on LPMs is suggested in L-DOPA-induced dyskinesia when the motor and facial nucleus are activated. Full article

Tigecycline (TGC), a third-generation tetracycline, is characterized by a more potent and broad antibacterial activity, and the ability to overcome different mechanisms of tetracycline resistance. TGC has proven to be of value in treatment of multidrug-resistant infections, but therapy can be complicated by multiple dangerous side effects, including direct drug toxicity. Given that, a TGC immunodetection method has been developed for therapeutic drug monitoring to improve the safety and efficacy of therapy. The developed indirect competitive ELISA utilized TGC selective antibodies and group-specific antibodies interacting with selected coating TGC conjugates. Both assay systems showed high sensitivity (IC50) of 0.23 and 1.59 ng/mL, and LOD of 0.02 and 0.05 ng/mL, respectively. Satisfactory TGC recovery from the spiked blood serum of healthy volunteers was obtained in both assays and laid in the range of 81–102%. TGC concentrations measured in sera from COVID-19 patients with secondary bacterial infections were mutually confirmed by ELISA based on the other antibody–antigen interaction and showed good agreement (R² = 0.966). A TGC pharmacokinetic (PK) study conducted in three critically ill patients proved the suitability of the test to analyze the therapeutic concentrations of TGC. Significant inter-individual PK variability revealed in this limited group supports therapeutic monitoring of TGC in individual patients and application of the test for population pharmacokinetic modelling. Full article

Extracellular adenosine 5′-triphosphate (ATP) is one of the best-known and frequently studied neurotransmitters. Its broad spectrum of biological activity is conditioned by the activation of purinergic receptors, including the P2X2 receptor. The P2X2 receptor is present in the central and peripheral nervous system of many species, including laboratory animals, domestic animals, and primates. However, the distribution of the P2X2 receptor in the nervous system of the domestic pig, a species increasingly used as an experimental model, is as yet unknown. Therefore, this study aimed to determine the presence of the P2X2 receptor in the neurons of the enteric nervous system (ENS) of the pig small intestine (duodenum, jejunum, and ileum) by the immunofluorescence method. In addition, the chemical code of P2X2-immunoreactive (IR) ENS neurons of the porcine small intestine was analysed by determining the coexistence of selected neuropeptides, i.e., vasoactive intestinal polypeptide (VIP), substance P (sP), and galanin. P2X2-IR neurons were present in the myenteric plexus (MP), outer submucosal plexus (OSP), and inner submucosal plexus (ISP) of all sections of the small intestine (duodenum, jejunum, and ileum). From 44.78 ± 2.24% (duodenum) to 63.74 ± 2.67% (ileum) of MP neurons were P2X2-IR. The corresponding ranges in the OSP ranged from 44.84 ± 1.43% (in the duodenum) to 53.53 ± 1.21% (in the jejunum), and in the ISP, from 53.10 ± 0.97% (duodenum) to 60.57 ± 2.24% (ileum). Immunofluorescence staining revealed the presence of P2X2-IR/galanin-IR and P2X2-IR/VIP-IR neurons in the MP, OSP, and ISP of the sections of the small intestine. The presence of sP was not detected in the P2X2-IR neurons of any ganglia tested in the ENS. Our results indicate for the first time that the P2X2 receptor is present in the MP, ISP, and OSP neurons of all small intestinal segments in pigs, which may suggest that its activation influences the action of the small intestine. Moreover, there is a likely functional interaction between P2X2 receptors and galanin or VIP, but not sP, in the ENS of the porcine small intestine. Full article

Brain aging involves regional alterations of specific cellular subpopulations in the human hippocampus: a network hub for memory consolidation. The present study investigates whether age, sex, education years, and the concentration of neuropathological and inflammatory proteins influence neuronal-type marker expression in the elderly hippocampus. We analyzed the digital images (1 µm/pixel) of postmortem hippocampal sections from 19 non-demented individuals (from 78 to 99 years). This material was obtained from the “Aging Dementia and TBI Study” open database. Brain samples were processed through in situ hybridization (ISH) for the immunodetection of VGLUT1 (glutamatergic transporter) and GAT1 (GABAergic transporter) and mRNAs and Luminex protein quantifications. After image acquisition, we delineated the dentate gyrus, CA 3/2, and CA1 hippocampal subdivisions. Then, we estimated the area fraction in which the ISH markers were expressed. Increased VGLUT1 was observed in multiple hippocampal subfields at late ages. This glutamatergic marker is positively correlated with beta-amyloid and tau proteins and negatively correlated with interleukin-7 levels. Additionally, education years are positively correlated with GAT1 in the hippocampus of elderly women. This GABAergic marker expression is associated with interferon-gamma and brain-derived neurotrophic factor levels. These associations can help to explain how hippocampal sub-regions and neurotransmitter systems undergo distinct physiological changes during normal aging. Full article

Prostate cancer is the second most common cancer diagnosed in men worldwide. Measuring the prostate-specific antigen (PSA) is regarded as essential during prostate cancer screening. Early diagnosis of this disease relapse after radical prostatectomy requires extremely sensitive methods. This research presents an approach to development of an ultrasensitive magnetic sandwich immunoassay, which demonstrates the limit of PSA detection in human serum of 19 pg/mL at a dynamic range exceeding 3.5 orders of concentration. Such attractive performance stems, inter alia, from the kinetic analysis of monoclonal antibodies (mAbs) against free PSA to select the mAbs exhibiting best kinetic characteristics and specificity. The analysis is carried out with a label-free multiplex spectral-correlation interferometry compatible with inexpensive single-use glass sensor chips. The high sensitivity of developed PSA immunoassay is due to electronic quantification of magnetic nanolabels functionalized by the selected mAbs and three-dimension porous filters used as an extended solid phase. The assay is promising for PSA monitoring after radical prostatectomy. The proposed versatile approach can be applied for the rational design of highly sensitive tests for detection of other analytes in many fields, including in vitro diagnostics, veterinary, food safety, etc. Full article

Tau hyperphosphorylation has been linked directly to the formation of toxic neurofibrillary tangles (NFTs) in tauopathies, however, prior to NFT formation, the sequence of pathological events involving tau phosphorylation remains unclear. Here, the effect of glycogen synthase kinase 3β (GSK3β) on tau pathology was examined independently for each step of transcellular propagation; namely, tau intracellular aggregation, release, cellular uptake and seeding activity. We find that overexpression of GSK3β-induced phosphorylated 0N4R tau led to a higher level of tau oligomerization in SH-SY5Y neuroblastoma cells than wild type 0N4R, as determined by several orthogonal assays. Interestingly, the presence of GSK3β also enhanced tau release. Further, we demonstrated that cells endocytosed more monomeric tau protein when pre-phosphorylated by GSK3β. Using an extracellular vesicle (EVs)-assisted tau neuronal delivery system, we show that exosomal GSK3β-phosphorylated tau, when added to differentiated SH-SY5Y cells, induced more efficient tau transfer, showing much higher total tau levels and increased tau aggregate formation as compared to wild type exosomal tau. The role of a primary tau phosphorylation site targeted by microtubule-affinity regulating kinases (MARKs), Ser262, was tested by pseudo-phosphorylation using site-directed mutagenesis to aspartate (S262D). S262D tau overexpression significantly enhanced tau release and intracellular tau accumulation, which were concurrent with the increase of pathological states of tau, as determined by immunodetection. Importantly, phosphorylation-induced tau accumulation was augmented by co-transfecting S262D tau with GSK3β, suggesting a possible interplay between Ser262 phosphorylation and GSK3β activity in tau pathology. Lastly, we found that pre-treatment of cells with amyloid-β (Aβ) further tau phosphorylation and accumulation when Ser262 pre-phosphorylation was present, suggesting that S262 may be a primary mediator of Aβ-induced tau toxicity. These findings provide a potential therapeutic target for treating tau-related disorders by targeting specific phospho-tau isoforms and further elucidate the GSK3β-mediated pathological seeding mechanisms. Full article

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods. Full article