Thermal Denaturation Research Articles

Effect mechanism of capsaicin and dihydrocapsaicin in chili on the oxidative stability of myoglobin in duck meat.

Myoglobin (Mb) in duck meat is commonly over-oxidized when heated at high temperatures, which may worsen the color of the meat. Enhancing the oxidative stability of Mb is essential for improving the color of duck meat. Capsaicin and dihydrocapsaicin (CA-DI) in chili exhibit antioxidant properties. This study investigated the effects of CA-DI on the structure and oxidative damage of Mb by fluorescence spectroscopy, differential scanning calorimetry analysis and particle size in duck meat during heat treatment. When the ratio of CA-DI to Mb was 10:1 g kg-1 and heat-treated for 36 min, oxymyoglobin significantly increased, and metmyoglobin significantly decreased compared with the control group (P < 0.05). In parallel, the carbonyl content of Mb in the CA-DI group decreased by 43.40 ± 0.10%, the sulfhydryl content increased by 188 ± 0.21%, and the free radical scavenging activity of Mb was significantly enhanced (P < 0.05). Moreover, the addition of CA-DI resulted in a significant decrease in the particle size of the Mb surface (P < 0.05). When the ratio of CA-DI to Mb was 10:1 g kg-1, CA-DI enhanced the thermal stability and significantly increased the thermal denaturation temperature of Mb. The molecular docking results indicated that hydrophobic interactions and hydrogen bonds were involved in the binding of CA-DI to Mb. CA-DI could combine with Mb and improve the oxidation stability of Mb in duck meat. This suggested that CA-DI could be a potential natural antioxidant that improves the color of meat products. © 2024 Society of Chemical Industry.

Role of N1-Domain, Linker, N2-Domain, and Latch in the Binding Activity and Stability of the Collagen-Binding Domain for the Collagen-Binding Protein Cbm from Streptococcus mutans

A special type of Streptococcus mutans expressing collagen-binding proteins (CBPs), Cnm, and Cbm, on the cell surface has been shown to be highly pathogenic. It is believed that S. mutans with CBPs that has entered the blood vessel attaches to collagen molecules exposed from the damaged blood vessel, inhibiting aggregation by platelets and increasing bleeding. Therefore, it is crucial to understand the molecular characteristic features of CBPs to protect against and cure S. mutans-related diseases. In this work, we highlighted the Cbm/collagen-binding domain (CBD) and examined its binding ability and thermal stability using its domain/region exchange variants in more detail. The CBD comprises the N1-domain, a linker, N2-domain, and a latch (N1–N2~), where the latch interacts with the N1-domain to form a β-sheet. The collagen-binding activity of the Cbm/CBD domain/region exchange variants was investigated using ELISA. Binding assays demonstrated that the N-domain_linker_N-domain composition is necessary for collagen binding as previously reported, newly that the latch is involved in binding through the β-sheet with the N1-domain when the N1-domain is present at the N-terminal position, and that the N2-domain is particularly important for collagen binding at both the N- and C-terminal positions. Thermal denaturation experiments newly revealed that the linker and latch bound to the N-domain contribute to N-domain stabilization but have no effect on the N-domain_linker_N-domain molecule, which contains two N-domains. It has also been shown that the N-terminal N2-domain destabilizes the N-domain_linker_N-domain structure. The results of this study will contribute to the rapid detection of CBP, development of CBP-targeted therapies, and application of CBPs to protein engineering using their collagen-binding ability.

Architecture and regulation of filamentous human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

Potassium Chloride as an Effective Alternative to Sodium Chloride in Delaying the Thermal Aggregation of Liquid Whole Egg.

The potential of potassium chloride (KCl) to be used as a substitute for sodium chloride (NaCl) was studied by monitoring the effects of salt treatment on thermal behavior, aggregation kinetics, rheological properties, and protein conformational changes. The results show that the addition of KCl can improve solubility, reduce turbidity and particle size, and positively influence rheological parameters such as apparent viscosity, consistency coefficient (K value), and fluidity index (n). These changes indicate delayed thermal denaturation. In addition, KCl decreased the content of β-sheet and random coil structures and increased the content of α-helix and β-turn structures. The optimal results were obtained with 2% KCl addition, leading to an increase in Tp up to 85.09 °C. The correlation results showed that Tp was positively correlated with solubility, α-helix and β-turn but negatively correlated with ΔH, turbidity, β-sheet and random coil. Overall, compared to NaCl, 2% KCl is more effective in delaying the thermal aggregation of LWE, and these findings lay a solid theoretical foundation for the study of sodium substitutes in heat-resistant liquid egg products.

The effects of nontoxic bio-based substances (egg white) on the performance and passivation of ZnO nanorods arrays-based light emitting devices

An innovative approach is proposed to passivate the existing defects from metal oxide semiconductors by functionalizing nontoxic bio-based substances. As a demonstration, we synthesized zinc oxide nanorods (ZnO NRs) using a hydrothermal method and incorporated chicken egg white (albumen) as a passivator to the defects. X-ray diffraction analysis of ZnO NRs shows enhanced quality and crystallinity features after incorporating albumen. XPS measurements were performed not only to introduce the chemical bonding between the albumen and the bare ZnO NRs but also specifically provide evidence of successful capping and defect passivation to the surface layer of ZnO NRs. It was observed that when the albumen was annealed, it formed sulfhydryl groups and disulfide bonds (which created disulfide bridges) from the chemical reaction in irreversible thermal denaturation. Steady-state photoluminescence of ZnO NRs showed two emission bands, i.e. near band-edge emission (NBE) and deep-level emission (DL). The NBE is significantly improved as compared to DL emission after capping and annealing the albumen, while the quenching of DL emission confirmed the reduced defects arising from the surface of ZnO NRs. The advantages and enhanced characteristics of the albumen-capped ZnO NRs led to fabricating a stable and highly efficient light-emitting device. This work opens the great potential of utilizing nontoxic and low-cost biomaterials in passivating the defects of metal oxide nanomaterials for the development of bio-inspired and stable optoelectronic devices.

Exploring the molecular basis of tucatinib interaction with human serum albumin: A spectroscopic and computational analysis

This study delves into the kinetics of interaction among anticancer drug tucatinib (TCT) and human serum albumin (HSA), a pivotal protein that transports substances in the bloodstream of humans. Employing biophysical techniques alongside in-silico approaches, the TCT-HSA complex formation is substantiated. This binding interaction is unequivocally confirmed by the inverse relationship between temperature and Stern-Volmer constant (KSV) and the hyperchromicity in UV–visible spectra. The binding constant (Kb) value (5.34 x 104 M−1) indicates a moderate affinity between TCT and HSA, with thermodynamic analysis pointing towards stabilization through hydrogen bonds and hydrophobic and van der Waals interactions. Moreover, despite causing alterations in the vicinity of Tyr and Trp amino acids, the introduction of TCT to HSA confers a substantial protective effect against the thermal denaturation of the protein. Circular dichroism unveils a decrease in the content of alpha helices upon TCT binding. At the same time, CD thermal analysis hints at a marginal increase in the melting temperature of HSA, indicating heightened stability at elevated temperatures. Molecular docking and competitive displacement assays confirm that the preferred binding site for TCT is subdomain IIA (Sudlow's site I) of HSA. Molecular dynamics simulations emphasize the stability of the TCT-HSA complex. This work sheds light on a detailed characterization of the HSA-TCT interaction, elucidating binding mechanisms and their consequential impact on HSA structure and stability.

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Physicochemical and foam properties of ovalbumin-carboxymethylcellulose mixtures after mild heat treatment: Comparison of electrostatic repulsion and attraction

The effects of electrostatic repulsion (pH6.5) and attraction (pH4.6) between biopolymers on the physicochemical and foam properties of ovalbumin (OVA)/carboxymethylcellulose (CMC) mixtures subjected to mild heat treatment (60–75 °C) were comparatively investigated. It was found that electrostatic binding between OVA and CMC at pH 4.6 resulted in an increase in the peak temperature of thermal denaturation (Td) of OVA from 71.3 °C to 74.4 °C, while no significant effect was observed at pH 6.5. Thus, 60 and 65 °C below Td were chosen to further investigate the effects of heating on the degree of thermal aggregation, protein conformation and foam properties of OVA/CMC mixtures. Confocal laser scanning microscopy observations and rheological results indicated that electrostatic binding between OVA and CMC at pH 4.6 significantly reduced the degree of protein thermal aggregation, while on the contrary, electrostatic repulsion between proteins and polysaccharides at pH 6.5 promoted OVA thermal aggregation. Raman spectroscopic characterisation indicated that mild heat treatment mainly led to a change in the microenvironment polarity of OVA tyrosine and tryptophan residues, as well as an increase in the content of random coil in the amide III band irrespective of pH. Moreover, after heating at 65 °C for 20 min, The foaming ability of the OVA/CMC mixtures at pH 6.5 and 4.6 increased by approximately 32% and 36% respectively compared to pure OVA. These findings have a positive value for enhancing the heat sterilisation strength of egg white proteins.

Design and Experimental Study of a Drilling Tool for Mars Rock Sampling

Martian rocks contain crucial information about the genesis of Mars and the historical evolution of Martian climate change. Consequently, extracting and examining Martian rocks are pivotal in advancing our comprehensive understanding of the red planet. However, the current core drill string is prone to wear and tear, and the samples are susceptible to thermal denaturation. To address these challenges, we introduce two novel types of drill bits, the conical straight junk slot and the conical spiral junk slot, both employing impregnated diamond as the primary material. Comparative experiments were meticulously conducted to evaluate the influence of different junk configurations on drilling parameters, including speed, abrasion resistance, drilling force loading, and sample temperature rise. The findings unequivocally demonstrate the superior performance of the spiral junk slot. Furthermore, simulations were performed to examine the drilling process on basalt using a fixed configuration drill bit, validating the occurrence of the sample temperature rise. The research presented in this paper offers valuable programmatic references and essential data support for future Martian rock coring drilling missions.

Research on improving the structural stability of surimi 3D printing through laser cooking techniques

At present, research on 3D printing technology for meat protein food is still in its early stages, and improving the molding stability and accuracy of products has always been a key challenge. Here, we take surimi protein as an example and achieve its localized thermal gelation quickly and precisely by incorporating laser sintering technology. And based on the thermal denaturation and solidification of proteins during the printing process, the structural instability problem of meat protein food 3D printing is solved. In this study, we used a CO2 laser with a power of 50 W to locally heat cure surimi during the printing process. The results show that under the conditions of 1 mm laser spot diameter, 500 mm/s scanning speed and 0.25 mm scanning interval, the shape accuracy and gel strength of surimi products with 1.5 mm layer thickness are significantly improved, and this combination parameter shows the best effect in the experiment. This study provides a theoretical basis and reference for real-time solidification in meat protein food printing.

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Temperature-Responsive Self-Assembly Nanochaperone Protects Green Fluorescent Proteins from Thermal Denaturation

Protein products perform important roles in the biochemistry field, but the thermal inactivation of proteins will increase the difficulty of their transport, storage and application. Therefore, improving the thermal stability of proteins has become a thorny challenge. Natural molecular chaperones can efficiently improve the resistance of proteins to environmental stimuli by reversible supramolecular assembly with proteins. Inspired by this machine, herein we designed a nanochaperone (nChap) with thermo-responsive amphiphilic surfaces that can prevent thermal denaturation and facilitate refolding of green fluorescent proteins (GFPs). By mimicking the hydrophobic microregion of natural chaperones, this nChap can effectively capture free GFPs and hide them into surface confined spaces, thereby shielding exposed hydrophobic sites of GFPs and preventing their irreversible thermal aggregation. When the heat stimulation disappeared, the thermosensitive segments of the nChaps underwent the hydrophilic transition, which provided suitable microenvironments for GFPs refolding. More importantly, nChaps could also actively adsorb to the surface of immobilized GFPs at high temperatures and realize the satisfactory dissociation of the nChap-protein complex upon cooling, which exhibited excellent chaperone-like activity. This work provides significant insights for understanding and developing strategies to improve protein stability.

Mechanism underlying the tenderness evolution of stir-fried pork slices with heating rate revealed by infrared thermal imaging assistance

This study aimed to explore the mechanism of cooking intensity on the tenderness of stir-fried pork slices from the perspective of the changes in temperature distribution. Infrared thermal imaging was used to monitor the distribution of temperature. Results showed that the high-level heat (HH) treatment could improve tenderness. When the center temperature increased to 100 °C, the shear force of samples from the low-level heat (LH) group increased by around 3-fold, and HH reduced this upward trend. This result was mainly attributed to the shorter heating time undergone by the HH-treated samples compared to the LH treatment, which resulted in less structural shrinkage and faster passing through the protein denaturation interval of the samples. These changes alleviated temperature fluctuations caused by water loss. This explanation could be confirmed by the results of T2 relaxation time and Fourier transform-infrared spectroscopy (FT-IR). However, the LH treatment caused a slower rise in oil temperature due to more moisture migration, which required the samples to undergo longer thermal denaturation, leading to a deterioration in tenderness. Moreover, histological analysis revealed that the greater integrity of endomysium in the HH group inhibited water loss and oil absorption, which contributed to obtain low-fat meat products with higher tenderness. This study provides support for the industrialization of traditional pork cuisines using oil as the heating medium.

Solid Phase Synthesis and TAR RNA-Binding Activity of Nucleopeptides Containing Nucleobases Linked to the Side Chains via 1,4-Linked-1,2,3-triazole.

Nucleopeptides (NPs) represent synthetic polymers created by attaching nucleobases to the side chains of amino acid residues within peptides. These compounds amalgamate the characteristics of peptides and nucleic acids, showcasing a unique ability to recognize RNA structures. In this study, we present the design and synthesis of Fmoc-protected nucleobase amino acids (1,4-TzlNBAs) and a new class of NPs, where canonical nucleobases are affixed to the side chain of L-homoalanine (Hal) through a 1,4-linked-1,2,3-triazole (HalTzl). Fmoc-protected 1,4-TzlNBAs suitable for HalTzl synthesis were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation of Fmoc-L-azidohomoalanine (Fmoc-Aha) and N1- or N9-propargylated nucleobases or their derivatives. Following this, two trinucleopeptides, HalTzlAAA and HalTzlAGA, and the hexanucleopeptide HalTzlTCCCAG, designed to complement bulge and outer loop structures of TAR (trans-activation response element) RNA HIV-1, were synthesized using the classical solid-phase peptide synthesis (SPPS) protocol. The binding between HalTzls and fluorescently labeled 5'-(FAM(6))-TAR UCU and UUU mutant was characterized using circular dichroism (CD) and fluorescence spectroscopy. CD results confirmed the binding of HalTzls to TAR RNA, which was evident by a decrease in ellipticity band intensity around 265 nm during complexation. CD thermal denaturation studies indicated a relatively modest effect of complexation on the stability of TAR RNA structure. The binding of HalTzls at an equimolar ratio only marginally increased the melting temperature (Tm) of the TAR RNA structure, with an increment of less than 2 °C in most cases. Fluorescence spectroscopy revealed that HalTzlAAA and HalTzlAGA, complementary to UUU or UCU bulges, respectively, exhibited disparate affinities for the TAR RNA structure (with Kd ≈ 30 and 256 µM, respectively). Hexamer HalTzlTCCCAG, binding to the outer loop of TARUCU, demonstrated a moderate affinity with Kd ≈ 38 µM. This study demonstrates that newly designed HalTzls effectively bind the TAR RNA structure, presenting a potential new class of RNA binders and may be a promising scaffold for the development of a new class of antiviral drugs.

Effect of vegetable oils on the thermal gel properties of PSE-like chicken breast meat protein isolate-based emulsion gels

To enhance the gel properties of PSE (pale, soft, and exudative)-like chicken meat protein isolate (PPI), the effect of peanut, corn, soybean, and sunflower oils on the gel properties of PPI emulsion gels was investigated. Vegetable oils improved emulsion stability and gel strength and enhanced viscosity and elasticity. The gel strength of the PPI–sunflower oil emulsion gel increased by 163.30 %. The thermal denaturation temperature and enthalpy values were increased. They decreased the particle size of PPI emulsion (P < 0.05) and changed the three-dimensional network structure of PPI emulsion gels from reticular to sheet with a smooth surface and pore-reduced lamellar. They elevated the content of immobile water PPI emulsion gels, decreased the α-helix and β-turn, and increased the β-sheet and random coil. Vegetable oil improved the gel properties of PPI in the following order: sunflower oil > soybean oil > corn oil ≈ peanut oil > control group.

Discovery of bioactive natural products of microbial origin as inhibitors of the PD-1/PD-L1 protein-protein interaction

The PD-1/PD-L1 protein-protein interaction (PPI) controls an adaptive immune resistance mechanism exerted by tumor cells to evade immune responses. The large-molecule nature of current commercial monoclonal antibodies against this PPI hampers their effectiveness by limiting tumor penetration and inducing severe immune-related side effects. Synthetic small-molecule inhibitors may overcome such limitations and have demonstrated promising clinical translation, but their design is challenging. Microbial natural products (NPs) are a source of small molecules with vast chemical diversity that have proved anti-tumoral activities, but which immunotherapeutic properties as PD-1/PD-L1 inhibitors had remained uncharacterized so far. Here, we have developed the first cell-based PD-1/PD-L1 blockade reporter assay to screen NPs libraries. In this study, 6000 microbial extracts of maximum biosynthetic diversity were screened. A secondary metabolite called alpha-cyclopiazonic acid (α-CPA) of a bioactive fungal extract was confirmed as a new PD-1/PD-L1 inhibitor with low micromolar range in the cellular assay and in an additional cell-free competitive assay. Thermal denaturation experiments with PD-1 confirmed that the mechanism of inhibition is based on its stabilization upon binding to α-CPA. The identification of α-CPA as a novel PD-1 stabilizer proves the unprecedented resolution of this methodology at capturing specific PD-1/PD-L1 PPI inhibitors from chemically diverse NP libraries.

Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ1: S258-T295; JlpAΔ2: Q332-A354) and assessed the Hsp90α-binding affinity–kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residues Q332-A354 are of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ2 to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.

THE EFFECT OF LOW HEATING TEMPERATURES ON CASEIN WHEY PROTEINS

In the work, a comparative analysis of the fractional composition and resistance to thermal denaturation of proteins of two samples of casein wheys was carried out: the laboratory one, which was obtained at a temperature of 18°C, and the production one – at a temperature of 46°C. In both cases, wheys were separated after isoelectric precipitation of casein complex proteins with hydrochloric acid. Express electrophoresis in a polyacrylamide gel showed an identical qualitative composition of major and minor proteins fractions in both samples. Also, no differences were found in the relative content of the main protein fractions of wheys – β-lactoglobulin (β-lg), α-lactalbumin (α-la), serum albumin (BSA) and immunoglobulins (Ig). To compare resistance to heat denaturation, samples of laboratory and production wheys were heated and held for 5 min at temperatures from 60°C to 100°C with an interval of 5°C. The precipitate of denatured proteins was isolated by centrifugation. The concentration of proteins in the supernatant was determined spectrophotometrically and the qualitative and quantitative composition of the main protein fractions was analysed by express electrophoresis. As a result, it was established that the concentration of undenatured protein in both wheys does not coincide in the temperature range of 70–90°C. Laboratory whey proteins were more resistant to heat denaturation. Moreover, these differences can be detected only with short-term (5 min) thermal denaturation. Long-term high-temperature heating (30 min) does not allow them to be detected. Electrophoretic analysis of the relative content of the main protein fractions showed that changes in stability occur due to two fractions – β-lg and BSA. The relative concentration of these fractions in the undenatured residue from production whey is ˂ 3%, and in the residue from laboratory whey &gt; 8% for BSA and &gt; 24% for β-lg when heated to 90°C for 5 min. Long-term exposure of both wheys at 20°C before investigating their resistance to thermal denaturation may indicate that some of the changes in the stability of production whey proteins caused by preheating to 46°C are irreversible.

Vaccine Based on Recombinant Fusion Protein Combining Hepatitis B Virus PreS with SARS-CoV-2 Wild-Type- and Omicron-Derived Receptor Binding Domain Strongly Induces Omicron-Neutralizing Antibodies in a Murine Model.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum-hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Understanding Antisense Oligonucleotide Efficiency in Inhibiting Prokaryotic Gene Expression.

Oligonucleotides offer a unique opportunity for sequence specific regulation of gene expression in bacteria. A fundamental question to address is the choice of oligonucleotide, given the large number of options available. Different modifications varying in RNA binding affinities and cellular uptake are available but no comprehensive comparisons have been performed. Herein, the efficiency of blocking expression of β-galactosidase (β-Gal) in E. coli was evaluated utilizing different antisense oligomers (ASOs). Fluorescein (FAM)-labeled oligomers were used to understand their differences in bacterial uptake. Flow cytometry analysis revealed significant differences in uptake, with high fluorescence seen in cells treated with FAM-labeled peptidic nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO) and phosphorothioate (PS) oligomers, and low fluorescence observed in cells treated with phosphodiester (PO) oligomers. Thermal denaturation (Tm) of oligomer:RNA duplexes and isothermal titration calorimetry (ITC) studies reveal that ASO binding to target RNA demonstrates a good correlation between Tm and Kd values. There was no correlation between Kd values and reduction of β-Gal activity in bacterial cells. However, cell-free translation assays demonstrated a direct relationship between Kd values and inhibition of gene expression by antisense oligomers, with tight binding oligomers such as LNA being the most efficient. Membrane active compounds such as polymyxin B and A22 further improved the cellular uptake of FAM-PNA and FAM-PS oligomers in wild-type E. coli cells. PNA and PMO were most effective in cellular uptake and reducing β-Gal activity as compared to oligomers with PS or those with PO linkages. Overall, cell uptake of the oligomers is shown as the key determinant in predicting their differences in bacterial antisense inhibition, and the RNA affinity is the key determinant in inhibition of gene expression in cell free systems.