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This study explored the intrinsic relationship between the quality traits and protein-related property changes during the solar drying of shrimps (Penaeus vannamei). During drying, the shrimp exhibited a gradual decline in L*a*b* values and a notable increase in the hardness and chewiness. Proteins were degraded and oxidized. Especially, the trichloroacetic acid-soluble peptide and carbonyl contents increased, whereas the total sulfhydryl content decreased. The proportions of different types of intramolecular bonds were significantly changed. The ionic and hydrogen bonds greatly decreased to 10.72% and 9.05% and the hydrophobic and disulfide bonds notably increased to 19.38% and 28.19%, indicating the changes in the spatial structure of the protein and its denaturation during the drying process. The Pearson's correlation analysis showed that protein degradation and denaturation greatly affected the textural properties and protein oxidation caused color changes. The result of this work provides a theoretical support for improving the quality of shrimp products.

Drying, as a critical step in the production of air-dried beef, has a direct impact on the quality of the final product. Innovatively, a composite system incorporating contact ultrasound (CU) and infrared radiation (IR) as auxiliary measures within a hot air drying (HAD) framework was built in this research, and the effects of these techniques on the drying kinetics, protein denaturation, and moisture transformation of air-dried beef were investigated. In comparison to HAD treatment, the integrated CU and IR (CU-IRD) system displayed marked enhancements in heat and moisture transport efficiency, thereby saving 36.84% of time expenditure and contributing favorably to the improved moisture distribution of the end-product. This was mainly ascribed to the denaturation of myosin induced by IR thermal effect and the micro-channel produced by CU sponge effect, thus increasing T2 relaxation time and the proportion of free water. In conclusion, the composite system solved the problem of surface hardening and reduces hardness and chewiness of air-dried beef by 40.42% and 45.25% respectively, but inevitably increased the energy burden by 41.60%.

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Background: Capparis cartilaginea Decne.(C. cartilaginea Decne.) a medicinally important plant that belongs to the family Capparaceae. Traditionally the fruit, leaf, and root have been believed to show anti-inflammatory effects and can also be used as agents. Purpose: The objective of this study is to investigate the chemical compositions, in-vitro antioxidant, anti-cancer and anti-inflammatory activities of the ethanolic extract of Capparis cartilaginea Decne. leaves. Study methods: The chemical compounds were investigated by Gas Chromatography–Mass Spectrometry (GC–MS) analysis. Ferric-bipyridine reducing capacity of total antioxidants (FBRC), Ferric reducing antioxidant power (FRAP) were utilized to determine antioxidant activity. The cytotoxic activity was evaluated using a 3-[4,5-dimethylthiazole- 2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Additionally, in vitro anti-inflammatory activity of the ethanolic extract of C. cartilaginea Decne. leaves using COX1 and protein denaturation methods. Results: The major twenty- four bioactive compounds from fifty compounds were identified including alkaloids, flavonoids, phenols, fatty acids, and carotenes. Pleiocarpamine was the major constituent of the extract (73.07 %). Additionally, in vitro anti-inflammatory activity of the ethanolic extract of C. cartilaginea Decne. leaves using COX1 and protein denaturation methods showed a strong effect with IC50 values of 60.23 and 17.67 µg/mL respectively, compared to Indomethacin and Diclofenac as a standard anti-inflammatory drug.; while FBRC and FRAP were utilized to determine antioxidant activity slowed high antioxidants capacity with values 0.056 ± 0.001and 0.03 ± 0.00196 Ascorbic acid equivalent/g of extract (AAE) and DPPH radical scavenging method show IC50 value 10 ± 1 %. Evaluation of the cytotoxic activity against three human cancer cell lines colon carcinoma cells HCT-116, Breast carcinoma cells MCF-7, and human muscle Rhabdomyosarcoma RD cell line using MTT assay showed a significant effect with IC50 values 59.58 ± 1.5, 39.14 ± 3.09, and 102 ± 8.75 µg/mL, compared to Vinblastine Sulfate as a standard cytotoxic drug. Yemeni C. cartilaginea Decne. showed a higher effect on MCF-7 compared to Iranian C. cartilaginea Decne. as well as established a higher antioxidant activity than that extracts obtained from Jordan. Conclusion: Ethanolic leaves extract of C. cartilaginea Decne. have the potential for development as therapeutic agents for cytotoxicity on certain cancer cells following further investigation. C. cartilaginea Decne. leaves that could have medicinal values and application in industries, were used to design the drug for the treatment of various types of inflammatory diseases.

The water distribution have a profound influence on meat quality, and proteins play a critical role in water distribution. The water distribution detected with proton NMR and its relationship with protein related properties were investigated. Three populations of water were detected: bound water (T21, P21), immobilized water (T22, P22), and free water (T23, P23). The decreased T22 and T23 indicated an increase in water-holding capacity in both muscles from 3 days of aging. The P22 in cattle was higher than that in yak and the P23 in cattle was lower than that in yak, suggesting that cattle exhibited a greater water-holding capacity compared to yak. Moreover, postmortem aging affected muscle protein oxidation, denaturation, and degradation. Correlation analysis suggested that protein oxidation and denaturation caused muscle water loss and protein degradation could allow the muscle to retain water. It provides a basis for the optimization of quality of meat and products.

Background: Rheumatoid arthritis (RA) is an inflammation of joints with increased cellularity of synovial tissue. Allopathic drugs possess several adverse effects, which have led to increase in the utilization of herbal medicines. Polyherbal emulgel resolves the bioavailability issue associated with hydrophobic drugs and can be used effectively in the treatment of RA. Objectives: The present study aimed at the formulation of polyherbal emulgel, and evaluation of in vitro anti-inflammatory activity and in vivo antiarthritic activity. Methods: Seven emulgels F-1 to F-7 were optimally formulated. In vitro anti-inflammatory activity was determined using protein denaturation method employing Diclofenac sodium as the standard. In antiarthritic study Complete Freund's Adjuvant (CFA) model was used. The various parameters were assessed, like paw volume, body weight, hematological parameters, antioxidant parameters, Rheumatic factor (RF), and histopathological study of ankle joint. Results: F-4 and F-7 were found to be optimized formulations as compared to other formulations. The in vitro anti-inflammatory activity was found to be highest in F-4 with IC50 7.74 and F-7 with IC50 8.87 in comparison with Diclofenac sodium having IC50 57.0. Both formulations F-7 and F-4 showed a significant reduction in paw volume and normalization of body weights. The formulation F-7 even showed more potent antiarthritic activity than F-4 by decreasing white blood cells (WBC), lymphocytes, increasing packed cell volume (PCV), neutrophils, superoxide dismutase (SOD), catalase and decreasing malondialdehyde (MDA) levels in serum. This was further confirmed by histopathological study. Conclusion: As an anti-inflammatory agent, this newly developed emulgel was found to possess more therapeutic efficacy than commercially available diclofenac sodium.

Noscapine hydrochloride (benzyl-isoquinoline antitussive alkaloid) is an opium derivative and generally used as a cough suppressant. Numerous studies on noscapine hydrochloride have reported that it has potent anti-inflammatory activity. However, the mechanisms by which it exerts an anti-inflammatory function is not well understood. Protein denaturation is the primary step that leads to the organ destruction and permanent arthritic disability. The above-mentioned facts provided the ground to plan this study using different in-vitro and in-vivo approaches. RT-qPCR and ELISA assays were used to assess the inflammatory markers related to protein denaturation in complete adjuvant persuaded rheumatism in Sprague - Dawley rats. The results were collected as paw volume and body weight changes, arthritic scoring and serum antioxidant enzymes assays. These findings demonstrated that all doses of noscapine hydrochloride (10, 20 and 40 mg/kg) studied in this study, significantly (p

Background: Oldenlandia corymbosa and Oldenlandia umbellata are well-known medicinal plants in various traditional systems of medicine and used to treat bronchitis, asthma, tuberculosis, constipation, and leprosy. We evaluated the phytochemical content, antioxidant potential, enzyme inhibitory action, anti-inflammatory, and antibacterial activities of the whole plant extracts of O. corymbosa and O. umbellata due to their therapeutic importance. Methods: The phytochemical analysis includes qualitative analysis and quantification of total phenolics, flavonoids, tannins, flavonols, proanthocyanidins, and vitamin E. Enzyme inhibitory assays were studied using α-amylase and α-glucosidase enzymes and anti-inflammatory activity was determined by egg albumin denaturation assay. Antioxidant potential was estimated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethyl benzothiozoline-6-sulfonic acid) diammonium salt (ABTS), ferric reducing antioxidant power (FRAP), hydroxyl radical, superoxide, nitric oxide, metal chelating, and phosphomolybdenum assays. Minimum inhibitory concentrations of extracts were studied against ten bacterial strains by the microdilution method. Results: The aqueous extract of O.umbellata showed a higher extract yield (5.0%). The highest amount of total phenolics and flavonoids were observed in the ethanolic extract of O.umbellata (77.23±0.46 mg GAE/g) and O.corymbosa (19.01±0.26 mg GAE/g). Ethanolic extract of O.umbellata (IC50 of 31.71±0.32 µg/mL) and aqueous extract of O.corymbosa (30.53±1.83 µg/mL) showed notable α-amylase inhibitory effect and petroleum ether extract of both plants observed good inhibition against α-glucosidase enzyme. Likewise, petroleum ether extract of O.umbellata (IC50 of 57.16±1.88 µg/mL) showed a moderate anti-inflammatory effect. In all extracts, considerable antioxidant potential was recorded specially with aqueous extract in the nitric oxide scavenging assay with an IC50 of 40.72±1.25 µg/mL. Ethanolic extract of O.umbellata and petroleum ether extract of O.corymbosa (MIC of 56.25 µg/mL) showed good antibiotic potency in lower concentrations which proves these two valuable herb's potential in herbal preparations. Conclusions: The results of the study provide a basis for further studies aiming to evaluate the possibilities of using the O. umbellata and O. corymbosa as natural sources of antioxidants, enzyme inhibitory, and antibacterial agents. Further studies could be directed to exploit these plants in experimental animal models as potential pharmaceutical agents.

The development of novel and sustainable food products, such as cheese- and meat analogues, requires a better understanding of the use of less refined ingredients. We investigated the distribution of water between the protein and starch phase of doughs and heat-induced gels made from air-classified faba bean fractions by developing a method suited for investigation of such multi-component ingredients. The moisture contents of the protein and starch phases in the dough were determined using a method based on partial sorption isotherms of mixed doughs of protein- and starch-rich fractions at high water activity. Water content of the protein phase is higher than that of the starch phase in dough, showing that protein takes up more water than starch at room temperature. Also, the moisture content of the protein phase in the gels was calculated using a model based on the denaturation temperature of legumin. From the experiments and the modelling, it became evident that the moisture content of the protein phase in the gel is lower than the moisture content of the protein phase in the dough, showing the importance of considering moisture migration from the protein to the starch during heating.

Blue light with a wavelength of 400–470 nm is the composition of the visible light. However, in recent years, blue light contributed the most significance to light pollution due to the artificial light at night. Previously, we have demonstrated that the Asian citrus psyllid (ACP), Diaphorina citri, an important pest in citrus production, has significant positive phototaxis with a light-emitting diode light of 400 nm. In this study, ACP with positive phototactic behavior to 400 nm light (PH) and non-phototactic behavior to 400 nm light (NP) were collected, individually. Transcriptome dynamics of head tissues of PH and NP groups were captured by using RNA-sequencing technology, respectively. Forty-three to 46 million clean reads with high-quality values were obtained, and 1773 differential expressed genes (DEGs) were detected. Compared with the NP group, there were 841 up-regulated DEGs and 932 down-regulated DEGs in the PH group. Eight pathways were significantly enriched in the PH group in the KEGG database, while 43 up-regulated pathways and 25 down-regulated pathways were significantly enriched in the PH group in the GO database. The DGE approach was reliable validated by real time quantitative PCR. Results indicated that the blue light acted as an abiotic stress causing physiological and biochemical responses such as oxidative stress, protein denaturation, inflammation and tumor development in ACPs. Additionally, the light was absorbed by photoreceptors of ACPs, and converted into electrical signal to regulate neuromodulation. This study provides basic information for understanding the molecular mechanisms of ACP in response to blue light and provides a reference for further studies to elucidate phototactic behavior.

In addition to the well-known monomeric and polymeric forms of actin there is another unique thermodynamically stable state of this protein, called "inactivated actin" (I-actin). I-actin is formed at moderate concentration of a denaturant, release of Ca 2+ ions and/or ATP, or after heating. This state is a monodisperse associate and it has the same spectral characteristics regardless of the method of preparation. The interest in I-actin arises from the discovery of similar-sized short oligomers of actin in the cell nucleus, which structurally differ from polymeric actin. In this work, we investigated the intramolecular mobility of I-actin using the time-resolved anisotropy method. Our findings indicate that its tryptophan residues participate in structural oscillations, although their correlation time is significantly longer than that of native actin. Using the dynamic light scattering, we demonstrated that I-actin obtained by heating possesses the same dimensions as I-actin in 1.8 M GdnHCl. Using the fluorescent probe ANS, it was shown that I-actin has a unique structure with hydrophobic pockets on the surface and tryptophan residues in the polar internal regions of the structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We aimed to create high-moisture plant-based meat alternatives (HPMAs) that closely mimic the fibrous structure found in conventional meat. To achieve this goal, we focused on the impact of modulating the heating and cooling die temperatures during the extrusion process on the structural properties of HPMAs. The optimal temperatures for the heating and cooling dies were determined to be 160 and 50 °C, respectively, following a comprehensive analysis of various quality characteristics. These included assessments of cutting strength, texturization degree, moisture content, and microstructure. Analyzing the correlation coefficients between heating and cooling die temperatures and the quality characteristics of HPMAs revealed that modulating the cooling die temperature has a greater impact on quality characteristics than modulating the heating die temperature. The quality characteristics of HPMAs were also compared with those of boiled and sous-vide cooked chicken breasts. The optimal HPMA exhibited the most similar fibrous macro- and micro-structure to sous-vide cooked chicken breast. Consequently, our study highlights the importance of modulating the heating die and cooling die temperatures, among various process parameters of high-moisture extrusion cooking, which is crucial for producing HPMAs closely resembling conventional meat., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pickering emulsions were co-stabilized by nanoliposome (NL) and thermally denatured ovalbumin (DOVA) based on the induction of OVA with strong particle characteristics through thermal denaturation. DOVA-NL particles were spherical and their sizes were mainly distributed between 50 and 100 nm. The surface tension and interfacial tension of DOVA-NL were significantly reduced, and the surface hydrophobicity, amphiphilicity and free -SH content of DOVA were enhanced after complexation with NL. The content of α-helix and β-sheet in DOVA decreased, whereas the content of β-turn and random coil increased after complexation with NL. Hydrophobic interactions, hydrogen bonding and electrostatic forces played a vital role in the interactions between NL and DOVA, leading to conformational changes in DOVA. The number of binding sites between NL and DOVA was more than one, and the interaction between NL and DOVA was exothermic and spontaneous. The emulsification index showed that DOVA-NL-stabilized Pickering emulsions (DNPE) were significantly more stable than DOVA-stabilized emulsions. DOVA-NL particles adsorbed at the oil-water interface and the droplet size of DNPE was smaller than that of DOVA-stabilized emulsions. This study suggests that it may be an effective strategy to improve the stability of Pickering emulsions through co-stabilization with NL and DOVA., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Physicochemical, proteomics, and bioinformatics analyses were conducted to investigate protein profiles in Trichiurus haumela under frozen (120 d) and chilled (6 d) storage. Springiness, chewiness, myofibrillar active sulfhydryl content, and Ca2+-ATPase activity significantly decreased, suggesting that cold stress altered muscle proteins. Compared with fresh hairtail (FH), 66 common differentially abundant proteins (DAPs) had lower abundances in chilled (3 d; CSH) and frozen (120 d; FSH) hairtail, including myosin binding proteins, filamins, actinin, troponin, and muscle-restricted coiled-coil protein. Gene Ontology (GO) annotation showed DAPs were mainly involved in cellular process, cellular anatomical entity, intracellular, and binding items. Eukaryotic orthologous group (KOG) analysis revealed that changes in cytoskeleton and energy production and conversion functions dominated during cold storage, degrading the myofibril and connective tissue structures and the physicochemical performance of muscle tissues. This study presents deep insights into the protein alternation mechanisms in hairtail muscle under cold stress.

The objective of this study was to investigate the influence of meat temperature on moisture loss, muscle water properties, and protein profiles in broiler pectoralis major with the severe woody breast (WB) condition. Broiler breast samples were collected from a commercial plant and sorted into normal, WB, and pale, soft, and exudative (PSE). Temperature treatments included 23°C, 40°C, 53°C, 57°C, 68°C, and 90°C based on denaturation of major muscle protein types during heating. Moisture loss was estimated with weight changes, water properties were measured with time-domain nuclear magnetic resonance measurements, and protein profiles were determined with SDS electrophoresis gel. There were no differences in moisture loss between 3 groups at meat temperature 23°C, 40°C, and 57°C. Moisture loss of WB samples was greater than normal and PSE at either 68°C or 90°C; however, it was the least at 53°C. Only close changing trend was noted between the intramyofibrillar water (T21) reduction and moisture loss. The extramyofibrillar (T22) water content and reduction in WB meat during heating were consistent greater, and electrophoretic profiles differed among 3 muscle conditions. Data suggest that greater reductions in intramyofibrillar and extramyofibrillar water are responsible for the increased moisture loss in WB meat at higher temperature.

Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

For irreversible denaturation transitions such as those exhibited by monoclonal antibodies, differential scanning calorimetry provides the denaturation temperature, T m , the rate of denaturation at T m , and the activation energy at T m . These three quantities are essential but not sufficient for an accurate extrapolation of the rate of denaturation to temperatures of 25 °C and below. We have observed that the activation energy is not constant but temperature dependent due to the existence of an activation heat capacity, C p,a . It is shown in this paper that a model that incorporates C p,a is able to account for previous observations like, for example, that increasing the T m does not always improve the stability at low temperatures; that some antibodies exhibit lower stabilities at 5 °C than at 25 °C; or that low temperature stabilities do not follow the rank order derived from T m values. Most importantly, the activation heat capacity model is able to reproduce time dependent stabilities measured by size exclusion chromatography at low temperatures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In this study, we examined the possibility of using industrial microwave processing to enhance the gelling properties and reduce the starch digestibility of mung bean flour (MBF). MBF (12.6 % moisture) was microwaved at a power of 6 W/g to different final temperatures (100-130 °C), and then its structural and functional properties were characterized. The microwave treatment had little impact on the crystalline structure or amylose content of the starch, but it roughened the starch granule surfaces and decreased the short-range ordered structure and degree of branching. In addition, the extent of mung bean protein denaturation caused by the microwave treatment depended on the final temperature. Slightly denaturing the proteins (100 °C) did not affect the nature of the gels (protein phase dispersed in a starch phase) but the gel network became more compact. Moderately denaturing the proteins (110-120 °C) led to more compact and homogeneous starch-protein double network gels. Excessive protein denaturation (130 °C) caused the gel structure to become more heterogeneous. As a result, the facilitated tangles between starch chains by more linear starch molecules after debranching, and the protein network produced by moderate protein denaturation led to the formation of stronger gel and the improvement of plasticity during large deformation (large amplitude oscillatory shear-LAOS). Starch recrystallization, lipid complexion, and protein network retard starch digestion in the MBF gels. In conclusion, an industrial microwave treatment improved the gelling and digestive properties of MBF, and Lissajous curve has good adaptability in characterizing the viscoelasticity of gels under large deformations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis → trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked at 55 °C (B-55) and 85 °C (B-85) showed an increase of β-sheet content by 3.2 % and a decrease of α-helix content by 3.3 %, indicating that proteins were gradually unfolded while oleosomes remained intact. The protein resisted thermal denaturation during secondary heating, and soymilks were stable as reflected by a small d 3,2 (0.4 μm). However, raw soymilk from soybeans baked at 115 °C (B-115), steamed for 1 min (ST-1) and 5 min (ST-5) presented oleosomes destruction and lipids aggregates. The proteins were coated around the oil aggregates. The β-turn content from soybeans steamed for 10 min (ST-10) increased by 9.5 %, with a dense network where the OBs were tightly wrapped, indicating the serious protein denaturation. As a result, the soymilks B-115 or steamed ones were unstable as evidenced by the serious protein aggregation and larger d 3,2 (5.65-12.48 μm). Furthermore, the soymilks were graininess and the protein digestion was delayed due to the formation of insoluble protein aggregates. The flavor and early-stage lipid digestion of soymilk from steamed soybeans was improved owing to lipid release., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study investigates the viscoelastic behavior, gelling properties, and structural characteristics of Deccan hemp seed protein (DHSP) to overcome limitations in its application in food formulations. Small amplitude oscillatory shear measurements were employed to investigate the impact of protein concentration, pH, ionic concentration, and temperature on DHSP's rheological features. The study revealed that the 20 % protein dispersion had the highest storage modulus (G') and yield stress at 63.96 ± 0.23 Pa and 0.61 Pa, respectively. DHSP dispersion exhibited pseudo-plastic behavior across various conditions. The gelling performance was higher at pH 4 and 8 and at ionic concentration in the range of 0.1 M - 0.5 M. Gelation time and temperature were observed from the temperature ramp test. Structural characterizations, including fluorescence spectroscopy, circular dichroism spectra, FTIR spectra, SEM, AFM images, zeta potential analysis, and DSC, provided insights into DHSP's tertiary and secondary conformation, surface characteristics, and thermal properties. Notably, the study highlighted DHSP's exceptional rheological properties, making it a promising gelling material for the food and nutraceutical industries. The findings also offer new insights into DHSP's structural characteristics, suggesting potential applications in food packaging and product development within the food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study evaluated the impact of high intensity ultrasound (HIU) on myofibrillar proteins (MP) from silver carp, and investigated the stabilizing effect of HIU-treated MP (UMP) on high internal phase emulsions (HIPEs). Ultrasonic cavitation induced protein denaturation by decreasing size and unfolding conformation, to expose more hydrophobic groups, particularly UMP at 390 W, showing the smallest particle size (181.71 nm) and most uniform distribution. These structural changes caused that UMP under 390 W exhibited the highest surface hydrophobicity, solubility (92.72 %) and emulsibility (115.98 m 2 /g and 70.4 min), all of which contributed to fabricating stable HIPEs with oil volume fraction up to 0.8. UMP-based HIPEs possessed tightly packed gel network and self-supporting appearance due to the adsorption of numerous proteins at the oil-water interface and the reduction of interfacial tension by protein reconfiguration. The larger interface coverage reinforced cross-linking between interfacial proteins, thus increasing the viscoelasticity and recoverability of HIPEs, also the resistance to centrifugal force, high temperature (90 °C, 30 min) and freeze-thaw cycles. These findings furnished insightful perspectives for MP deep processing through HIU, expanding the high-value application of UMP-based HIPEs in fat replacer, nutritional delivery system with high encapsulation content and novel 3D printing ink., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Several species of Bridelia have been used in the condition of pain & arthritis in Indian folk medicine. Present study revealed the preliminary phytochemical investigation and evaluation of analgesic, anti-inflammatory and anti-arthritic activity as well as underlying mechanism of bark of Bridelia retusa Spreng. (Euphorbiaceae). The bark was subjected to extraction using pet.ether, ethyl acetate and acetone. All the extracts were significantly inhibit abdominal writhings response and licking time in late phase of formalin test. Extracts could also significantly inhibit mean paw edema of rats induced by carrageenan & histamine at dose of 200 & 400 mg/kg, i.p. Test materials also showed significant dose dependent reduction in cotton pellet granuloma & acetic acid induced vascular permeability at 400 mg/kg. Oral administration of B. retusa fractions in CFA induced arthritic rats, physical, biochemical and hematological parameters observed in arthritic animals were altered significantly to near normal condition. The maximum paw edema inhibition at day 21 was observed at 400 mg/kg. It also proved significant protection against protein denaturation & RBC membrane damage. The GC-MS analysis of EA extract revealed the presence of β-sitosterol, stigmasterol, lupeol and friedelin (Pentacyclic triterpenoid). Therefore present study has demonstrated the analgesic; anti-inflammatory and anti-arthritic activities of B. retusa bark and suggested that the molecular membrane might be associated with inhibition of biochemical and hematological parameters. Overall bioactive profile of B. retusa used phytomedicine in future for inflammatory conditions.

Variations in pork quality impact consumer acceptance, and fibre type differences between muscles contribute to this variation. The aim was to investigate the influence of variations in muscle fibre types and protein denaturation peaks across four pork muscles and the influence of ageing and cooking temperature on longissimus quality traits. The longissimus, masseter, cutaneous trunci, and psoas major from 13 carcases were removed 1-day postmortem and subjected to 0- or 14-days ageing (d0, d14). Quality traits, protein denaturation peak temperature (DSC), fibre diameter and fibre type proportions were measured. Cook loss for longissimus was similar on d0 and d14, but was higher on d14 for masseter, cutaneous trunci, and psoas major. Warner-Bratzler shear force was highest, and ultimate pH was lowest, for longissimus, and similar among cutaneous trunci, masseter, and psoas major. Masseter had lowest L* and highest a* and longissimus and cutaneous trunci had highest L* and lowest a*. The DSC temperature peaks for longissimus occurred at lower temperatures relative to the other muscles. Fibre diameter was largest for type-IIb fibres relative to type-IIa and type-I. Longissimus and cutaneous trunci had predominantly type-IIb glycolytic (71%, 51% respectively), masseter had predominantly type-IIa intermediate (50%) and psoas major had predominantly type-I oxidative (48%) fibres. The glycolytic longissimus had the lowest DSC temperature peaks and the lowest quality meat. Masseter had the highest proportion of type-I fibres but was generally similar in quality traits to psoas major, and also similar to cutaneous trunci which had more glycolytic fibres than masseter., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Jacalin, the jackfruit seed lectin, exhibits high specificity for the tumor-specific T-antigen and is used in various biomedical and biotechnological applications. Here, we report biophysical studies on the thermal unfolding of jacalin and the effect of pH and temperature on its secondary structure. Differential scanning calorimetric (DSC) studies revealed that native jacalin unfolds at ∼60 °C and that carbohydrate binding stabilizes the protein structure. Circular dichroism spectroscopic studies indicated that the secondary structure of jacalin remains mostly unaffected over pH 2.0-9.0, whereas considerable changes were observed in the tertiary structure. DSC experiments demonstrated that jacalin exhibits two overlapping transitions between pH 2 and 5, which could be attributed to dissociation of the tetrameric protein into subunits and their unfolding. Interestingly, only one transition between pH 6 and 9 was observed, suggesting that the subunit dissociation and unfolding occur simultaneously. While quenching of the protein intrinsic fluorescence by acrylamide increased significantly upon carbohydrate binding, quenching by succinimide is essentially unaffected. We attribute this difference to increased exposure of Trp-123 in the α-chain as it is involved in carbohydrate binding. Both acrylamide and succinimide gave biphasic Stern-Volmer plots, consistent with differential accessibility of the two tryptophan residues of jacalin to them., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Musti J. Swamy reports financial support was provided by India Ministry of Science & Technology Department of Science and Technology. Musti J. Swamy reports financial support was provided by University of Hyderabad., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nanotechnology is an emerging technology that uses medicinal plants to extract nanoparticles for conventional applications. In the present investigation, the medical plant Tulsi (Ocimum sanctum) has used in the synthesis of cobalt (Co) nanoparticles in a cost-effective, feasible process. The efficiency of nanoparticles in removing methyl orange dye was evaluated by analyzing their applications in wastewater treatment. An analysis of the anti-inflammatory and anti-cancer properties of Tulsi-mediated Co nanoparticles was conducted to examine their medical application. Morphological analysis of Co nanoparticles showed that the synthesized nanoparticles were in crystal shape with a mean particle size of 110 nm. A batch adsorption study has shown that incubation periods of 5 h, pH 2, temperatures of 70 °C, and adsorbent dosage of 125 μg/mL are optimal for removing methyl orange dye from wastewater. To examine the anti-inflammatory properties of Tulsi-mediated Co nanoparticles, protein denaturation and nitric oxide scavenging assays were performed. The maximum anti-inflammatory response was recorded at a concentration of 250 μg/mL of Co nanoparticles. MTT assays against MDA-MB-231 human breast cancer cells were used to evaluate the anti-cancer properties of Co nanoparticles. This study investigates the economical extraction of Co nanoparticles from tulsi and its potential use in wastewater purification and biomedical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Rheumatoid arthritis (RA) is an inflammation of joints with increased cellularity of synovial tissue. Allopathic drugs possess several adverse effects, which have led to increase in the utilization of herbal medicines. Polyherbal emulgel resolves the bioavailability issue associated with hydrophobic drugs and can be used effectively in the treatment of RA., Objectives: The present study aimed at the formulation of polyherbal emulgel, and evaluation of in vitro anti-inflammatory activity and in vivo antiarthritic activity., Methods: Seven emulgels F-1 to F-7 were optimally formulated. In vitro anti-inflammatory activity was determined using protein denaturation method employing Diclofenac sodium as the standard. In antiarthritic study Complete Freund's Adjuvant (CFA) model was used. The various parameters were assessed, like paw volume, body weight, hematological parameters, antioxidant parameters, Rheumatic factor (RF), and histopathological study of ankle joint., Results: F-4 and F-7 were found to be optimized formulations as compared to other formulations. The in vitro anti-inflammatory activity was found to be highest in F-4 with IC 50 7.74 and F-7 with IC 50 8.87 in comparison with Diclofenac sodium having IC 50 57.0. Both formulations F-7 and F-4 showed a significant reduction in paw volume and normalization of body weights. The formulation F-7 even showed more potent antiarthritic activity than F-4 by decreasing white blood cells (WBC), lymphocytes, increasing packed cell volume (PCV), neutrophils, superoxide dismutase (SOD), catalase and decreasing malondialdehyde (MDA) levels in serum. This was further confirmed by histopathological study., Conclusion: As an anti-inflammatory agent, this newly developed emulgel was found to possess more therapeutic efficacy than commercially available diclofenac sodium., Competing Interests: Declaration of competing interest The authors declare no Conflict of Interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's β and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To study the influence of Cd(II) ions on denaturation kinetics of hen egg white lysozyme (HEWL) under thermal and acidic conditions, spontaneous Raman spectroscopy in conjunction with Thioflavin-T fluorescence, AFM imaging, far-UV circular dichroism spectroscopy, and transmittance assays was conducted. Four distinctive Raman spectral markers for protein tertiary and secondary structures were recorded to follow the kinetics of conformational transformation. Through comparing variations of these markers in the presence or absence of Cd(II) ions, Cd(II) ions show an ability to efficiently accelerate the disruption of tertiary structure, and meanwhile, to promote the direct formation of organized β-sheets from the uncoiling of α-helices by skipping intermediate random coils. More significantly, with the action of Cd(II) ions, the initially resulting oligomers with disordered structures tend to assemble into aggregates with random structures like gels more than amyloid fibrils, along with a so-called "off-pathway" denaturation pathway. Our results advance the in-depth understanding of corresponding ion-specific effects., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Xiaoguo Zhou reports financial support was provided by National Natural Science Foundation of China. Lei Xing reports financial support was provided by China Postdoctoral Science Foundation.]., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Enzyme function is governed by a complex network of conformational changes and internal dynamics, with the same getting more convoluted in the crowded cellular environment. Here, we have explored an intricate interplay amongst activity, structure, conformation, and dynamics of a multidomain enzyme, AK3L1 (UniProtKB: Q9UIJ7) in the crowded milieu. We have monitored changes in the enzyme landscape in response to the chemical denaturant, urea, under the influence of different concentrations of macromolecular crowders. Extensive experimental analyses using FRET-based domain displacement measurements, sub-nanosecond time scale local dynamics, and global structural changes, along with enzymatic activity studies, have been carried out to get deeper insights into the factors that may modulate the functional landscape of adenylate kinase (AK3L1). It was observed that AK3L1 gets activated at low urea concentrations, whereas higher urea concentrations unfold and thereby deactivate the enzyme. A sequential response of AK3L1 is observed towards external perturbation (urea) occurring through a series of well-defined steps. Incorporation of crowders not only shift the maximum activity of enzyme to a higher urea concentration, but also enhance domain compaction, as revealed by FRET studies. The modulation in enzyme activity and solvation dynamics acting as local response, precede global unfolding of the enzyme, indicating that the structural alterations around the active site are quite decoupled from the large amplitude global transitions., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Syntenin-1 is a multidomain protein containing a central tandem of two PDZ domains flanked by two unnamed domains. Previous structural and biophysical studies show that the two PDZ domains are functional both isolated and in tandem, occurring a gain in their respective binding affinities when joined through its natural short linker. To get insight into the molecular and energetic reasons of such a gain, here, the first thermodynamic characterization of the conformational equilibrium of Syntenin-1 is presented, with special focus on its PDZ domains. These studies include the thermal unfolding of the whole protein, the PDZ-tandem construct and the two isolated PDZ domains using circular dichroism, differential scanning fluorimetry and differential scanning calorimetry. The isolated PDZ domains show low stability (ΔG < 10 kJ·mol -1 ) and poor cooperativity compared to the PDZ-tandem, which shows higher stability (20-30 kJ·mol -1 ) and a fully cooperative behaviour, with energetics similar to that previously described for archetypical PDZ domains. The high-resolution structures suggest that this remarkable increase in cooperativity is associated to strong, water-mediated, interactions at the interface between the PDZ domains, associated to nine conserved hydration regions. The low T m value (45 °C), the anomalously high unfolding enthalpy (>400 kJ·mol -1 ), and native heat capacity values (above 40 kJ·K -1 ·mol -1 ), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics., Competing Interests: Declaration of competing interest The authors declare that they do not have conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cystatin superfamily members, by virtue of their thiol protease regulatory properties, show involvement in myriad physiological processes important for survival and well-being. The current study involves urea-induced denaturation of a novel variant of the cystatin superfamily, rai seed cystatin (RSC), employing a variety of biophysical assays in order to characterize different folding intermediates generated on unfolding. Urea as a denaturant presented the passage of RSC through a series of events resulting in the loss of RSC functional capability, accompanied by changes in the archetype at secondary and tertiary structural levels, as evident from protease inhibitory, UV absorption, and intrinsic fluorescence assays, respectively. ANS fluorescence also revealed routing of RSC through discrete multiple sub-states thus presenting the generation of intermediate states somewhat close to the pre-molten globule and/or molten globule forms of RSC. Furthermore, far-UV circular dichroism analysis revealed a concentration-dependent gradual loss in typical -helical RSC peaks, indicating a nearly 50 % loss in secondary structural elements around 5 M urea treatment. The study also reports the possible role of glycerol in the refolding and/or reactivation of the urea unfolded RSC form. Glycerol presented itself as a potent structural stabilizer as it assisted in the refolding and reactivation of the unfolded RSC in a dosage-dependent manner, concomitantly paving the way for unravelling the mechanistic approach involved in the phenomenon, which can facilitate future studies., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest in this work., (Copyright © 2023. Published by Elsevier B.V.)

Cryogenic electron microscopy (cryo-EM) is now one of the most powerful and widely used methods to determine high-resolution structures of macromolecules. A major bottleneck of cryo-EM is to prepare high-quality vitrified specimen, which still faces many practical challenges. During the conventional vitrification process, macromolecules tend to adsorb at the air-water interface (AWI), which is known unfriendly to biological samples. In this review, we outline the nature of AWI and the problems caused by it, such as unpredictable or uneven particle distribution, protein denaturation, dissociation of complex and preferential orientation. We review and discuss the approaches and underlying mechanisms to deal with AWI: 1) Additives, exemplified by detergents, forming a protective layer at AWI and thus preserving the native folds of target macromolecules. 2) Fast vitrification devices based on the idea to freeze in-solution macromolecules before their touching of AWI. 3) Thin layer of continuous supporting films to adsorb macromolecules, and when functionalized with affinity ligands, to specifically anchor the target particles away from the AWI. Among these supporting films, graphene, together with its derivatives, with negligible background noise and mechanical robustness, has emerged as a new generation of support. These strategies have been proven successful in various cases and enable us a better handling of the problems caused by the AWI in cryo-EM specimen preparation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The effect of pre-rigor temperature incubation on the activity and distribution in sarcoplasmic and myofibrillar fractions of calpains, and meat quality attributes was investigated. Porcine longissimus thoracis muscles were incubated pre-rigor at 14, 22, 30 and 38 °C to 6 h postmortem, followed by another 2 h incubation at 14 °C. Thereafter, muscles were stored at 2 °C for 1 or 4 days. With higher pre-rigor temperature, sarcoplasmic Ca 2+ concentration, purge loss and myofibril-bound calpain-1 content increased, while shear force declined. Water-holding capacity of isolated myofibrils was lower after pre-rigor incubation at 38 °C. Desmin and troponin T degradation, and myofibril fragmentation was greater upon incubation of isolated myofibrils with added Ca 2+ in the order 800 μM Ca 2+  > 40 μM Ca 2+  > no Ca 2+ , suggesting that calpain-1 and calpain-2 were associated to myofibrils and proteolytically active with sufficient Ca 2+ . Activity of myofibril-bound calpain-1 in muscle incubated pre-rigor at 22 and 30 °C were higher than when incubated at 14 and 38 °C. These results indicate that calpains translocate from the sarcoplasm onto myofibrils with higher pre-rigor temperature to 30 °C and the proteolytic potential of myofibril-associated calpains is thereby increased., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Thermal denaturation of human serum albumin has been the subject of many studies in recent decades, but the results of these studies are often conflicting and inconclusive. To clarify this, we combined different spectroscopic and calorimetric techniques and performed an in-depth analysis of the structural changes that occur during the thermal unfolding of different conformational forms of human serum albumin. Our results showed that the inconsistency of the results in the literature is related to the different quality of samples in different batches, methodological approaches and experimental conditions used in the studies. We confirmed that the presence of fatty acids (FAs) causes a more complex process of the thermal denaturation of human serum albumin. While the unfolding pathway of human serum albumin without FAs can be described by a two-step model, consisting of subsequent reversible and irreversible transitions, the thermal denaturation of human serum albumin with FAs appears to be a three-step process, consisting of a reversible step followed by two consecutive irreversible transitions., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chemotaxis Y (CheY), upon metal binding, displays a drastic alteration in its structure and stability. This premise prompted us to study the effect of crowding on the two conformationally distinct states of the same test protein. A comparative analysis on the structure and thermal stability in the presence and absence of the macromolecular crowder, ficoll, and its monomeric unit, sucrose, revealed a contrasting effect of ficoll on the apo and holo forms. In the presence of ficoll while the thermal stability (T m ) of the apo form is enhanced, the thermal stability of the holo form is reduced. The selective lowering of T m for the holo form in the combined presence of ficoll and sucrose and not in sucrose alone suggests that the contrasting effect is due to the macromolecular nature of ficoll. Since metal-protein interaction remains unperturbed in the presence of ficoll and Mg 2+ sequestration is ruled out in a systematic manner the alternative possibility for the exclusive reduction in the thermal stability of the holo form is the ficoll-induced modulation of the relative population of apo and holo forms of CheY., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Non-covalent intramolecular interactions play a key role in the protein folding process. Aminoacidic mutations or changes in physiological conditions such as pH and/or temperature variations can compromise intramolecular stability generating misfolding or unfolding proteins with consequent impairment of functionality and the triggering of pathological states. The intramolecular HINT scoring function recently implemented and validated, is proposed as a rapid and sensitive method for the evaluation of different conformational states characterizing destabilization processes. In this work, the stability of Transthyretin, whose denaturation is related to amyloid fibril formation, is evaluated by generating multiple structural mutated models under different pH conditions in comparison with experimental data. These results suggest that the HINT scoring function can be used for an accurate and rapid evaluation and computational prediction of the effects of structural changes on any protein system., Competing Interests: Declaration of competing interest None Declared., (Copyright © 2023. Published by Elsevier Ltd.)

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Blue light with a wavelength of 400-470 nm is the composition of the visible light. However, in recent years, blue light contributed the most significance to light pollution due to the artificial light at night. Previously, we have demonstrated that the Asian citrus psyllid (ACP), Diaphorina citri, an important pest in citrus production, has significant positive phototaxis with a light-emitting diode light of 400 nm. In this study, ACP with positive phototactic behavior to 400 nm light (PH) and non-phototactic behavior to 400 nm light (NP) were collected, individually. Transcriptome dynamics of head tissues of PH and NP groups were captured by using RNA-sequencing technology, respectively. Forty-three to 46 million clean reads with high-quality values were obtained, and 1773 differential expressed genes (DEGs) were detected. Compared with the NP group, there were 841 up-regulated DEGs and 932 down-regulated DEGs in the PH group. Eight pathways were significantly enriched in the PH group in the KEGG database, while 43 up-regulated pathways and 25 down-regulated pathways were significantly enriched in the PH group in the GO database. The DGE approach was reliable validated by real time quantitative PCR. Results indicated that the blue light acted as an abiotic stress causing physiological and biochemical responses such as oxidative stress, protein denaturation, inflammation and tumor development in ACPs. Additionally, the light was absorbed by photoreceptors of ACPs, and converted into electrical signal to regulate neuromodulation. This study provides basic information for understanding the molecular mechanisms of ACP in response to blue light and provides a reference for further studies to elucidate phototactic behavior., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Raman spectroscopy was employed to study the thermal denaturation of three different proteins, bovine serum albumin (BSA), lysozyme, ovalbumin; and the decomposition temperature of three amino acids, l-glutamine, l-cysteine, and l-alanine, all of them as lyophilized powders. All the Raman bands observed in the spectra obtained were recorded and analyzed at preset heating temperatures. The results obtained for either protein denaturation temperature T D and amino acid decomposition temperatures T M-dc , were compared with those measured by differential scanning calorimetry (DSC). The DSC and Raman results were additionally corroborated with a thermogravimetric analysis (TGA) for the case of proteins. This exercise indicated almost complete coincidence in the determination of these transition temperatures between the three techniques, evidencing the applicability of Raman spectroscopy in the study of denaturation and decomposition temperatures of proteins and amino acids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The phenomenon of foam-induced inactivation is a common challenge during foam fractionation of proteins. In this work, we attempted to use ultrasound to treat foam out of the foam fractionation column with the aim of minimizing the foam-induced protein inactivation using pepsin as a model protein. Firstly, the mechanisms by which ultrasound prevented the foam-induced pepsin inactivation during defoaming were explored. The results showed that ultrasound promoted the refolding of unfolded pepsin molecules at the gas-liquid interface to restore their activities during the desorption process from the interface and before dissolving into the foamate. Subsequently, the effects of ultrasonic power on the pepsin renaturation were analyzed, which revealed that the unfolding degree of pepsin in the foamate gradually decreased and then increased as the ultrasonic power increased. Correspondingly, the specific activity of pepsin in the foamate increased and then decreased. Finally, we explored the effects of defoaming efficiency, type of gas and pH. The results indicated that the decrease in defoaming efficiency and gas solubility in water facilitated the ultrasound-assisted pepsin renaturation in the pH range from 1.0 to 2.5. When air was applied in the foaming process, the specific activity of pepsin in the foamate with ultrasonic treatment was not significantly different from that in the feed solution at ultrasonic power of 400 W and pH 2.0. Collectively, these results indicate the ultrasonic treatment of foam effectively prevented the foam-induced pepsin inactivation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rui Li reports financial support was provided by Rizhao Science and Technology Bureau., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn 2+ ). Here we investigated the conformational stability of ageritin in the pH range 4.0-7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C. At pH 5.0 we calculated a value of 36 kJ mol -1 for the unfolding Gibbs energy at 25 °C. We also analysed the thermodynamic and catalytic behaviour of S-pyridylethylated form, obtained by alkylating the single Cys18 residue, which is predicted to bind Zn 2+ . We show that this form possesses the same activity and structure of ageritin, but lower stability. In fact, the corresponding values of 52 °C and 14 kJ mol -1 were found. Conservation of activity is consistent with the location of alkylation site on the opposite site of the catalytic site cleft. Inasmuch as Cys18 is part of a structurally stabilizing zinc-binding site, disrupted by cysteine alkylation, our results point to an important role of metal ions in ageritin stability., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from ice crystallization and the freezing-induced denaturation of myofibrillar proteins, the latter of which has, however, not received much research focus. This review discusses the relationship between myofibrillar protein denaturation and water-holding capacity of meat in freezing-thawing with the aim to improve the understanding the relative importance of protein denaturation in the formation of thaw loss. The contribution of decreased pH and high ionic strength in the unfrozen water in freezing is emphasized and we hypothesize that these two factors are causing protein denaturation and conformational changes within muscle fibers, and consequently loss of water-holding capacity. Slow freezing produces more thaw loss than fast freezing, and this is discussed here in relation to the impacts on myofibrillar protein denaturation induced by the freezing rate., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)