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Journal of the Science of Food and... Jun 2024Morden advanced analytical tools offer valuable information into the understanding of molecular mechanism of traditional food processing. Chopping temperature is...
BACKGROUND
Morden advanced analytical tools offer valuable information into the understanding of molecular mechanism of traditional food processing. Chopping temperature is well-known to affect the surimi gel quality of silver carp, but the detailed molecular mechanism is not very clear. In this study, a gel-based proteomics was performed on the extracted surimi proteins under different chopping temperatures (0, 5, 10, and 25 °C) along with other physicochemical characterization of surimi proteins and gels.
RESULTS
With increased chopping temperature, protein extractability (in 3% sodium chloride) generally decreased, while the extracted protein generally exhibited larger surface hydrophobicity, reduced intrinsic fluorescence intensity, lower sulfhydryl content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile of extracted protein showed a clear difference at 25 °C when compared with the other three temperatures, and more protein fragmentation occurred. Proteomic analysis of selected bands indicated that major myofibrillar proteins react differently with chopping temperatures, especially at 25 °C. The selected bands contained a variety of other proteins or their fragments, including adenosine triphosphate (ATP) synthase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate isomerase, heat shock protein, parvalbumin, collagen, and so forth. For the surimi gel, water-holding capacity and gel strength generally decreased with increased chopping temperature.
CONCLUSION
Our results suggested that chopping at 0-10 °C is acceptable for the production of silver carp surimi in terms of gel strength and water-holding capacity. However, a chopping temperature near 0 °C led to less protein oxidation and denaturation. The inferior gel quality at 25 °C is linked to a decreased concentration of extracted protein and degradation of major myofibrillar protein, the latter is likely crosslinked with sarcoplasmic proteins. © 2024 Society of Chemical Industry.
PubMed: 38860545
DOI: 10.1002/jsfa.13654 -
Protein Expression and Purification Jun 2024Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate,...
Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and Chlamydia. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in CT's developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.
PubMed: 38857716
DOI: 10.1016/j.pep.2024.106532 -
BioRxiv : the Preprint Server For... May 2024display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of...
display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing -(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.
PubMed: 38853888
DOI: 10.1101/2024.05.29.596443 -
International Journal of Biological... Jun 2024Glycyrrhiza glabra Linn (liquorice) has been widely used for therapeutic purposes to treat digestive disorders, immunomodulatory disorders, inflammatory disorders,...
Highly swellable, cytocompatible and biodegradeable guar gum-based hydrogel system for controlled release of bioactive components of liquorice (Glycyrrhiza glabra L.): Synthesis and evaluation.
Glycyrrhiza glabra Linn (liquorice) has been widely used for therapeutic purposes to treat digestive disorders, immunomodulatory disorders, inflammatory disorders, diabetes, viral infections, and cancer. Liquorice contains a wide variety of bioactive compounds, including glycyrrhizin, flavonoids, and terpenoids. Several factors compromise their therapeutic efficacy, such as poor pharmacokinetic profiles and physicochemical properties. Therefore, to improve its overall effectiveness, liquorice solid dispersion (LSD) was incorporated into biopolymer-based guar gum-grafted-2-acrylamido-2-methylpropane sulfonic acid (Guar gum-g-AMPS) hydrogels designed for controlled delivery via the oral route and characterized. The qualitative analysis of LSD revealed 51 compounds. Hydrogel structural properties were assessed for their effect on swelling and release. The highest swelling ratio (6413 %) and drug release (84.12 %) occurred at pH 1.2 compared to pH 7.4 (swelling ratio of 2721 % and drug release of 79.36 %) in 48 h. The hydrogels exhibited high porosity (84.23 %) and biodegradation (9.30 % in 7 days). In vitro hemolysis tests have demonstrated the compatibility of the hydrogel with blood. CCK-8 assay confirmed the biocompatibility of the synthesized hydrogel using osteoblasts and RIN-m5f cells. LSD exhibited good anti-inflammatory activity when loaded into hydrogels after being subjected to protein denaturation experiments. Moreover, LSD-loaded hydrogels have good antioxidant and antibacterial properties.
PubMed: 38852724
DOI: 10.1016/j.ijbiomac.2024.132825 -
Food Chemistry Jun 2024This study aimed to upcycle a byproduct of the edible oil industry, cold-pressed nettle seed meal (CPNSM), into a plant-based emulsifier, thereby increasing the...
This study aimed to upcycle a byproduct of the edible oil industry, cold-pressed nettle seed meal (CPNSM), into a plant-based emulsifier, thereby increasing the sustainability of the food system. The protein content of the nettle seed protein (NSP) powder was 48.3% with glutamic acid (16.6%), asparagine (10.7%), and arginine (9.7%) being the major amino acids. NSPs had a denaturation temperature of 66.6 °C and an isoelectric point of pH 4.3. They could be used as emulsifiers to form highly viscous coarse corn oil-in-water emulsions (10% oil, 4% NSP). Nevertheless, 10-fold diluted emulsions exhibited rapid creaming under different pH (2-9), salt (0-500 mM NaCl) and temperature (>40 °C) conditions, but they were relatively stable to aggregation. Our findings suggest that NSPs could be used as emulsifiers in highly viscous or gelled foods, like dressings, sauces, egg, cheese, or meat analogs.
PubMed: 38852455
DOI: 10.1016/j.foodchem.2024.139878 -
Pathology May 2024Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such...
Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.
PubMed: 38852040
DOI: 10.1016/j.pathol.2024.04.001 -
Chemistry & Biodiversity Jun 2024The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of...
The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.
PubMed: 38849308
DOI: 10.1002/cbdv.202401038 -
Journal of Food Science Jun 2024The present study investigated the effects of different deep-frying times and temperatures on the amylose content, crystal structure, thermodynamics, and other...
The present study investigated the effects of different deep-frying times and temperatures on the amylose content, crystal structure, thermodynamics, and other properties of deep-fried dough sticks. Results showed that the change of amylose content in deep-fried dough sticks during the deep-frying process was positively correlated with time and temperature. Moreover, the deep-frying process of deep-fried dough sticks was accompanied by the formation of starch-lipid complexes that led to the destruction of starch structure. The degreased sample and the oil sample had the same absorption peaks at 2854 and 1746 cm, respectively. The melting enthalpy (ΔH) of the starch-lipid complex decreased significantly. In addition, the viscosity of starch reduced as the deep-frying time and temperature increased. Furthermore, it was found that the effect of increasing deep-frying temperature was greater than that of time. PRACTICAL APPLICATION: As a popular deep-fried food, the main component of deep-fried dough sticks is starch. Starch gelatinization, protein denaturation, and interaction among components occurred during deep-frying. At present, there are few studies focusing on the properties of starch in deep-fried dough sticks in the real deep-frying system. Therefore, this study provided a theoretical basis for subsequent research by measuring the effects of different deep-frying conditions on the properties of starch in deep-fried dough sticks.
PubMed: 38847754
DOI: 10.1111/1750-3841.17152 -
Current Health Sciences Journal 2024Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed...
Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed to the release of free radicals by immune cells, prooxidants that can also cause protein alteration. This study was performed in order to assess the potential anti-inflammatory effect of two bee venom samples harvested from Apis mellifera. Free radical scavenging capacity was investigated using DPPH and ABTS.+ tests and protective effect on proteins through the inhibitory activity on thermal denaturation of albumin.
PubMed: 38846469
DOI: 10.12865/CHSJ.50.01.11 -
The Analyst Jun 2024Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1-1 μg per kg body weight, has attracted much attention regarding criminal and terroristic misuse...
Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1-1 μg per kg body weight, has attracted much attention regarding criminal and terroristic misuse over the past decade. Therefore, developing a rapid detection method for abrin toxin is of great significance in the field of biosecurity. In this study, based on the specific dissociation method of an immobilized enzyme reactor, the trypsin immobilized reactor FeO@CTS-GA-Try was prepared to replace free trypsin, and the immobilized enzyme digestion process was systematically investigated and optimized by using bovine serum albumin as the simulant of abrin. After 5 min one-step denaturation and reduction, a satisfactory peptide number and coverage were yielded with only 15 s assisted by an ultrasound probe to identify model proteins. Subsequently, abrin was rapidly digested using the established method, resulting in a stable and highly reproducible characteristic peptide number of 39, which can be analyzed by nanoelectrospray ionization coupled with high-resolution mass spectrometry. With the acquisition mode of full MS scan coupled with PRM, not only MS spectroscopy of total abrin peptides but also the corresponding MS/MS spectroscopy of specific abrin peptides can achieve the characteristic detection of abrin toxin and its different isoforms in less than 10 minutes, with high repeatability. This assay provides a universal platform and has great potential for the development of on-site detection and rapid mass spectrometric analysis techniques for macromolecular protein toxins and can further be applied to the integrated detection of chemical and biological agents.
PubMed: 38845587
DOI: 10.1039/d4an00406j