Search Results (31)

β-parvalbumin (β-PV) is the primary fish allergen responsible for most allergic reactions in individuals sensitive to fish. To ensure food safety, a sandwich-based magnetic immunoassay was developed using maleimide-functionalized magnetic beads (NH-MBs). Specific anti-β-PV antibodies were immobilized on these MBs, and a screen-printed carbon electrode was employed as the electrochemical transducer. A linear concentration range from 10 to 1000 ng/mL, a limit of detection of 1.8 ng/mL, and a limit of quantification of 7.1 ng/mL were achieved. Nineteen commercial food samples were analyzed to assess the potential of the sensor for routine use in food quality control. Important factors such as protein source and food processing (e.g., boiling, grilling, and frying) and preservation (e.g., in oil, and vacuum) were evaluated. The validated results confer the usefulness of the assay for food quality control. Full article

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX. Full article

For decades, amyloid β-peptide (Aβ) misfolding aggregates with β-sheet structures have been linked to the occurrence and advancement of Alzheimer’s disease (AD) development and progression. As a result, modulating the misfolding mode of Aβ has been regarded as an important anti-amyloid protein misfolding strategy. A polyoxometalate based on {Co(H₂O)₄}²⁺ complex and [P₂Mo₅O₂₃]⁶⁻ fragments, K₈{[Co(H₂O)₄][HP₂Mo₅O₂₃]₂}·8H₂O (abbreviated as CoPM), has been synthesized and structurally characterized using elemental analysis, single-crystal X-ray diffraction (SXRD), IR, UV spectra, bond valence sums (Σs) calculation, and powder XRD (PXRD). CoPM’s primary component, as revealed by structural analysis, is a nanoscale polyoxoanion made of [Co(H₂O)₄]²⁺ sandwiched between two [P₂Mo₅O₂₃]⁶⁻ pieces. Notably, it is demonstrated that CoPM efficiently modulates Aβ aggregates’ β-sheet-rich conformation. Full article

Protein biomarkers in nasal secretions can be used as a measure to differentiate between allergies, airway diseases and infections for non-invasive diagnostics. The point-of-care quantification of biomarker levels using flow-based microarray facilitates precise and rapid diagnosis and displays the potential for targeted and effective treatment. For the first time, we developed a flow-based chemiluminescence sandwich microarray immunoassay (CL-SMIA) for the quantification of nasal interferon-beta (IFN-β) on the Microarray Chip Reader-Research (MCR-R). Polycarbonate foils are used as a cost-effective surface for immobilizing capture antibodies. By using a commercially available set of anti-human IFN-β antibodies, the CL-SMIA can be compared directly to an enzyme-linked immunosorbent assay (ELISA) performed in microtiter plates concerning the bioanalytical performance and economic issues. Pre-incubation of the sample with detection antibodies facilitates the lower consumption of detection antibodies, as this allows for a longer interaction time between the antibody and the biomarker. The direct injection of pre-incubated samples into the microarray chips eliminates the adsorption of proteins in the tubing as well as the contamination of the tubing and valves of the MCR-R with clinical samples. The small flow cell allows for a low sample volume of 50 μL. The limit of detection of 4.53 pg mL⁻¹ was slightly increased compared to a sandwich ELISA performed on microtiter plates which were 1.60 pg mL⁻¹. The possibility to perform the CL-SMIA in a multiplexed mode makes it a promising assay for the rapid and cost-effective non-invasive detection of biomarkers in nasal secretions. Full article

Background and Objectives: The Mediterranean diet’s bioactive components are suggested to strengthen the immune system and to exert anti-inflammatory actions. This study investigated the association between adherence to the Mediterranean diet with serum inflammatory factors, total antioxidant capacity, appetite, and symptoms of COVID-19 patients. Materials and Methods: This cross-sectional study was conducted among 600 Iranian COVID-19 patients selected by a simple random method. The ten-item Mediterranean diet adherence questionnaire was used to assess diet adherence. At the beginning of the study, 5 cc of blood was taken from all patients for measurement of serum interleukin 1β) IL-1β), tumor necrosis factor (TNF-α), malondialdehyde (MDA), high sensitivity C-reactive protein (hs-CRP) and total antioxidant capacity (TAC). A human ELISA kit with serial number 950.090.096 produced by the Diaclone Company was used to test this cytokine using the sandwich ELISA method. Results: One hundred and five patients presented a high adherence and 495 patients presented a low adherence to the Mediterranean diet. The incidence of fever, cough, diarrhea, taste changes, and pneumonia severity index were significantly lower in patients who adhered to the Mediterranean diet more than other patients. Serum levels of tumor necrosis factor (5.7 ± 2.1 vs. 6.9 ± 2.8 p = 0.02), interleukin 1 beta (3.2 ± 0.02 vs. 4.9 ± 0.01 p = 0.02), high-sensitivity C-reactive protein (17.08 ± 4.2 vs. 19.8 ± 2.5 p = 0.03), and malondialdehyde (5.7 ± 0.2 vs. 6.2 ± 0.3 p = 0.02) were significantly lower in patients who adhered more to the Mediterranean diet than other patients. Conclusion: The Mediterranean diet can improve the symptoms and elevated serum inflammatory factors in COVID-19 patients, so clinical trial studies are suggested to confirm this effect. Full article

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500–1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMT_HMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure—unlike other amyloidogenic proteins—formed without changes in the SMT_HMW secondary structure. SMT_HMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMT_HMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis. Full article

Consumption of aquatic food, including fish, accounts for 17% of animal protein intake. However, fish consumption might also result in several side-effects such as sneezing, swelling and anaphylaxis in sensitized consumers. Fish allergy is an immune reaction to allergenic proteins in the fish muscle, for instance parvalbumin (PV), considered the major fish allergen. In this study, we characterize PV in two economically important fish species for southern European aquaculture, namely gilthead seabream and European seabass, to understand its stability during in vitro digestion and fish processing. This information is crucial for future studies on the allergenicity of processed fish products. PVs were extracted from fish muscles, identified by mass spectrometry (MS), and detected by sandwich enzyme-linked immunosorbent assay (ELISA) after simulated digestion and various food processing treatments. Secondary structures were determined by circular dichroism (CD) after purification by anion exchange and gel filtration chromatography. In both species, PVs presented as α-helical and β-sheet structures, at room temperature, were shown to unfold at boiling temperatures. In European seabass, PV detectability decreased during the simulated digestion and after 240 min (intestinal phase) no detection was observed, while steaming showed a decrease (p < 0.05) in PVs detectability in comparison to raw muscle samples, for both species. Additionally, freezing (−20 °C) for up to 12 months continued to reduce the detectability of PV in tested processing techniques. We concluded that PVs from both species are susceptible to digestion and processing techniques such as steaming and freezing. Our study obtained preliminary results for further research on the allergenic potential of PV after digestion and processing. Full article

In this study, we analyze the occurrence of the unique structural motif, the 3β-corner, belonging to the Structural Classification of Proteins (SCOP) folds, in proteins of various origins. We further assess the structural and functional role of this motif as well as the clustering of the biological functions of proteins in which it occurs. It has been shown previously that the 3β-corner occurs with different probabilities in all beta proteins, alpha and beta proteins (α + β and α/β), and alpha classes occur most often in the composition of β-proteins. The 3β-corner is often found as a building block in protein structures, such as β-barrels, -sandwiches, and -sheets/-layers. Full article

At the first step of phage infection, the receptor-binding proteins (RBPs) such as tail fibers are responsible for recognizing specific host surface receptors. The proper folding and assembly of tail fibers usually requires a chaperone encoded by the phage genome. Despite extensive studies on phage structures, the molecular mechanism of phage tail fiber assembly remains largely unknown. Here, using a minimal myocyanophage, termed Pam3, isolated from Lake Chaohu, we demonstrate that the chaperone gp25 forms a stable complex with the tail fiber gp24 at a stoichiometry of 3:3. The 3.1-Å cryo-electron microscopy structure of this complex revealed an elongated structure with the gp25 trimer embracing the distal moieties of gp24 trimer at the center. Each gp24 subunit consists of three domains: the N-terminal α-helical domain required for docking to the baseplate, the tumor necrosis factor (TNF)-like and glycine-rich domains responsible for recognizing the host receptor. Each gp25 subunit consists of two domains: a non-conserved N-terminal β-sandwich domain that binds to the TNF-like and glycine-rich domains of the fiber, and a C-terminal α-helical domain that mediates trimerization/assembly of the fiber. Structural analysis enabled us to propose the assembly mechanism of phage tail fibers, in which the chaperone first protects the intertwined and repetitive distal moiety of each fiber subunit, further ensures the proper folding of these highly plastic structural elements, and eventually enables the formation of the trimeric fiber. These findings provide the structural basis for the design and engineering of phage fibers for biotechnological applications. Full article

Protein folding is a complicated phenomenon including various time scales (μs to several s), and various structural indices are required to analyze it. The methodologies used to study this phenomenon also have a wide variety and employ various experimental and computational techniques. Thus, a simple speculation does not serve to understand the folding mechanism of a protein. In the present review, we discuss the recent studies conducted by the author and their colleagues to decode amino acid sequences to obtain information on protein folding. We investigate globin-like proteins, ferredoxin-like fold proteins, IgG-like beta-sandwich fold proteins, lysozyme-like fold proteins and β-trefoil-like fold proteins. Our techniques are based on statistics relating to the inter-residue average distance, and our studies performed so far indicate that the information obtained from these analyses includes data on the protein folding mechanism. The relationships between our results and the actual protein folding phenomena are also discussed. Full article

Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants—Fusobacterium nucleatum and Enterococcus faecalis—as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/β-Catenin, TGF-β, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/β-Catenin and NF-κB signaling pathways: β-Catenin (p = 0.0068 *), LRP-5 (p = 0.0059 **), and LRP-6 (p = 0.0329 *), as well as NF-kB (p = 0.0034 **) and TRAF6 (p = 0.0285 *). These results suggest that oral bacteria can up- and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET. Full article

Galectins are multi-purpose effectors acting via interactions with distinct counterreceptors based on protein-glycan/protein recognition. These processes are emerging to involve several regions on the protein so that the availability of a detailed structural characterization of a full-length galectin is essential. We report here the first crystallographic information on the N-terminal extension of the carbohydrate recognition domain of rat galectin-5, which is precisely described as an N-tailed proto-type-like galectin. In the ligand-free protein, the three amino-acid stretch from Ser2 to Ser5 is revealed to form an extra β-strand (F0), and the residues from Thr6 to Asn12 are part of a loop protruding from strands S1 and F0. In the ligand-bound structure, amino acids Ser2–Tyr10 switch position and are aligned to the edge of the β-sandwich. Interestingly, the signal profile in our glycan array screening shows the sugar-binding site to preferentially accommodate the histo-blood-group B (type 2) tetrasaccharide and N-acetyllactosamine-based di- and oligomers. The crystal structures revealed the characteristically preformed structural organization around the central Trp77 of the CRD with involvement of the sequence signature’s amino acids in binding. Ligand binding was also characterized calorimetrically. The presented data shows that the N-terminal extension can adopt an ordered structure and shapes the hypothesis that a ligand-induced shift in the equilibrium between flexible and ordered conformers potentially acts as a molecular switch, enabling new contacts in this region. Full article

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC₃) and nitrogenized graphene (C₃N) sandwiched stripe (BC₃/C₃N/BC₃) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C₃N stripe than the BC₃ one, which leads to their capture, elongation, and confinement along the center C₃N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise. Full article

The declaration of lupine supplements is mandatory to avoid lupine allergy for sensitive individuals. However, reliable detection methods against lupine allergen remain critical to prevent the unintended consumption of allergen contaminated food. In this study, we have immunized an alpaca with lupine protein extracts and retrieved nanobodies (Nbs). Nevertheless, the target antigen has been recognized as Lup an 1, which has been classified as β-conglutin, and confirmed to connect with lupine allergy. After selection of the best Nb-pair, a sandwich enzyme-linked immunosorbent assay (ELISA) has been developed providing a linear range of 0.036–4.4 μg/mL with detection limit of 1.15 ng/mL. This immunoassay was confirmed by detecting the samples with spiked allergen, and a recovery from 86.25% to 108.45% with coefficient of variation (CV) less than 4.0% has been determined. Generally, this study demonstrated the selection of Nbs against allergen with crude protein content to develop the immunoassay for lupine surveillance in foods. Full article

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection. Full article