Protein Layer Research Articles

In vitro investigation of protein assembly by combined microscopy and infrared spectroscopy at the nanometer scale

The nanoscale structure and dynamics of proteins on surfaces has been extensively studied using various imaging techniques, such as transmission electron microscopy and atomic force microscopy (AFM) in liquid environments. These powerful imaging techniques, however, can potentially damage or perturb delicate biological material and do not provide chemical information, which prevents a fundamental understanding of the dynamic processes underlying their evolution under physiological conditions. Here, we use a platform developed in our laboratory that enables acquisition of infrared (IR) spectroscopy and AFM images of biological material in physiological liquids with nanometer resolution in a cell closed by atomically thin graphene membranes transparent to IR photons. In this work, we studied the self-assembly process of S-layer proteins at the graphene-aqueous solution interface. The graphene acts also as the membrane separating the solution containing the proteins and Ca2+ ions from the AFM tip, thus eliminating sample damage and contamination effects. The formation of S-layer protein lattices and their structural evolution was monitored by AFM and by recording the amide I and II IR absorption bands, which reveal the noncovalent interaction between proteins and their response to the environment, including ionic strength and solvation. Our measurement platform opens unique opportunities to study biological material and soft materials in general.

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans.

To investigate the molecular basis of biofilm formation in a recombinant lab strain of Deinococcus radiodurans with a plasmid harbouring gfp and kanR that acquired the biofilm-forming ability. Deinococcus radiodurans R1 is known as a nonbiofilm former bacterium and so far there are no reports on its biofilm-producing capabilities. In this study, we investigated the molecular basis of biofilm formation in a recombinant strain of D. radiodurans using classical biofilm assays, confocal laser scanning microscopy and real-time PCR. Biochemical analysis of D. radiodurans biofilm matrix revealed that it consisted predominantly of protein and carbohydrate complexes with a little amount of extracellular DNA (eDNA). Furthermore, studies showed that D. radiodurans biofilm formation was enhanced in the presence of 25 mM Ca2+ , which enhanced the exopolysaccharide and protein content in the biofilm matrix. Enzymatic treatments with proteinase K, alginate lyase and DNase I indicated the involvement of some proteinaceous components to be critical in the biofilm formation. RT-PCR studies showed that increased expression of a surface layer protein SlpA conferred the biofilm ability to D. radiodurans. Overexpression of SlpA in D. radiodurans conferred the biofilm formation ability to the bacterium, in which a partial role was also played by the recombinant plasmid pKG. It was also shown that the presence of Ca2+ in the growth medium enhanced SlpA production, thus improving biofilm stability and biofilm maturation of D. radiodurans. This study shows how biofilm formation can be augmented in D. radiodurans. The finding has implications for the development of D. radiodurans biofilm-based biotechnological applications.

Profiling of time-dependent human plasma protein adsorption on non-coated and heparin-coated oxygenator membranes.

Patients with severe lung diseases are highly dependent on lung support systems. Despite many improvements, long-term use is not possible, mainly because of the strong body defence reactions (e.g. coagulation, complement system, inflammation and cell activation). The systematic characterization of adsorbed proteins on the gas exchange membrane of the lung system over time can provide insights into the course of various defence reactions and identify possible targets for surface modifications. Using comprehensive mass spectrometry analyses of desorbed proteins, we were able to identify for the first time binding profiles of over 500 proteins over a period of six hours on non-coated and heparin-coated PMP hollow fiber membranes. We observed a higher degree of remodeling of the protein layer on the non-coated membrane than on the coated membrane. In general, there was a higher protein binding on the coated membrane with exception of proteins with a heparin-binding site. Focusing on the most important pathways showed that almost all coagulation factors bound in higher amounts to the non-coated membranes. Furthermore, we could show that the initiator proteins of the complement system bound stronger to the heparinized membranes, but the subsequently activated proteins bound stronger to the non-coated membranes, thus complement activation on heparinized surfaces is mainly due to the alternative complement pathway. Our results provide a comprehensive insight into plasma protein adsorption on oxygenator membranes over time and point to new ways to better understand the processes on the membranes and to develop new specific surface modifications.

In silico integrative analysis of multi-omics reveals regulatory layers for diurnal gene expression in mouse liver.

Diurnal oscillation persists throughout the body and plays an essential role in maintaining physiological homeostasis. Disruption of diurnal rhythm contributes to many diseases including type 2 diabetes. The regulatory mechanism of the transcription-translation feedback loop (TTFL) of core clock genes is well-established, while a systematic study across all regulatory layers of gene expression, including gene transcription, RNA translation, and DNA binding protein (DBP) activities, is still lacking. We comprehensively bioinformatics analyzed the rhythmicity of gene transcription, mature RNA abundance, protein abundance and DBP activity using publicly available omic-datasets from mouse livers. We found that the core clock genes, Bmal1 and Rev-erbα, persistently retained rhythmicity in all stages, which supported the essential rhythmic function along with the TTFL. Interestingly, there were many layer-specific rhythmic genes playing layer-specific rhythmic functions. The systematic analysis of gene transcription rate, RNA translation efficiency, and post-translation modification of DBP were incorporated to determine the potential mechanisms for layer-specific rhythmic genes. We observed the gene with rhythmic expression in both mature RNA and protein layers were largely due to relatively consistent translation rate. In addition, rhythmic translation rate induced the rhythms of protein whose mature RNA levels were not rhythmic. Further analysis revealed a phosphorylation-mediated and an enhancer RNA-mediated cycling regulation between the corresponding layers. This study presents a global view of the oscillating genes in multiple layers via a systematical analysis and indicates the complexity of regulatory mechanisms across different layers for further functional study.

Isolation and Characterization of Cell Envelope Fragments Comprising Archaeal S-Layer Proteins.

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive.

Lactobacillus buchneri S-layer protein-coated liposomes loaded with β-cyclodextrin–carvacrol inclusion complexes for the enhancement of antibacterial effect

This article describes the development of a novel liposome nanocarrier system. Carvacrol (Car) is first embedded in β-cyclodextrin (β-CD) by the freeze-drying method to form the β-cyclodextrin-carvacrol inclusion compound (β-CD–Car), and then β-CD–Car liposomes (β-CD–Car-LPs) and β-CD–Car liposomes coated with S-layer proteins (SLPs) from Lactobacillus buchneri 20023 (SLP/β-CD–Car-LPs) were prepared. The liposomes were characterized, and their stabilities, in vitro release characteristics, and antibacterial activities were investigated. Results showed that the fabricated liposome SLP/β-CD–Car-LPs was nanosized, oval and homogenous, with the particle size of 229.1 ± 6.81 nm, the polydispersity index of 0.139, and the zeta potential of 27.9 mV. Measurements based on Triton X-100 resistance indicated that the SLP-coated liposomes were more stable than naked liposomes. The in vitro release study results showed that the rate of release from SLP-coated liposomes was much lower than that from uncoated liposomes. The minimum inhibitory activity (MIC) of SLP/β-CD–Car-LPs (0.05 mg/mL) was 6.4 times higher than that of the free carvacrol (0.32 mg/mL) and was twice that of β-CD–Car-LPs (0.1 mg/mL). In general, the stability, antibacterial activity, and sustained release effect of β-CD–Car-LPs modified with SLPs were improved. Findings suggested that SLP-coated liposomes could be developed as a favorable delivery system for potential applications in the food industry.

Protein's Protein Corona: Nanoscale Size Evolution of Human Immunoglobulin G Aggregates Induced by Serum Albumin.

Nanoparticles are readily coated by proteins in biological systems. The protein layers on the nanoparticles, which are called the protein corona, influence the biological impacts of the nanoparticles, including internalization into cells and cytotoxicity. This study expands the scope of the nanoparticle's protein corona for exogenous artificial nanoparticles to that for exogenous proteinaceous nanoparticles. Specifically, this study addresses the formation of protein coronas on nanoscale human antibody aggregates with a radius of approximately 20-40 nm, where the antibody aggregates were induced by a pH shift from low to neutral pH. The size of the human immunoglobulin G (hIgG) aggregates grew to approximately 25 times the original size in the presence of human serum albumin (HSA). This size evolution was ascribed to the association of the hIgG aggregates, which was triggered by the formation of the hIgG aggregate's protein corona, i.e., protein's protein corona, consisting of the adsorbed HSA molecules. Because hIgG aggregate association was significantly reduced by the addition of 30-150 mM NaCl, it was attributed to electrostatic attraction, which was supported by molecular dynamics (MD) simulations. Currently, the use of antibodies as biopharmaceuticals is concerning because of undesired immune responses caused by antibody aggregates that are typically generated by a pH shift during the antibody purification process. The present findings suggest that nanoscale antibody aggregates form protein coronas induced by HSA and the resulting nanoscale antibody-HSA complexes are stable in blood containing approximately 150 mM salt ions, at least in terms of the size evolution. Mechanistic insights into protein corona formation on nanoscale antibody aggregates are useful for understanding the unintentional biological impacts of antibody drugs.

Human Vimentin Layers on Solid Substrates: Adsorption Kinetics and Corona Formation Investigations.

Adsorption kinetics of human vimentin on negatively charged substrates (mica, silica, and polymer particles) was analyzed using atomic force microscopy (AFM), quartz microbalance (QCM), and the laser doppler velocimetry (LDV) method. AFM studies realized under diffusion conditions proved that the adsorbed protein layer mainly consisted of aggregates in the form of compact tetramers and hexamers of a size equal to 11–12 nm. These results were consistent with vimentin adsorption kinetics under flow conditions investigated by QCM. It was established that vimentin aggregates efficiently adsorbed on the negatively charged silica sensor at pH 3.5 and 7.4, forming compact layers with the coverage reaching 3.5 mg m–2. Additionally, the formation of the vimentin corona at polymer particles was examined using the LDV method and interpreted in terms of the electrokinetic model. This allowed us to determine the zeta potential of the corona as a function of pH and the electrokinetic charge of aggregates, which was equal to −0.7 e nm–2 at pH 7.4 in a 10 mM NaCl solution. The anomalous adsorption of aggregates exhibiting an average negative charge on the negatively charged substrates was interpreted as a result of a heterogeneous charge distribution. These investigations confirmed that it is feasible to deposit stable vimentin layers both at planar substrates and at carrier particles with well-controlled coverage and zeta potential. They can be used for investigations of vimentin interactions with various ligands including receptors of the innate immune system, immunoglobulins, bacterial virulence factors, and spike proteins of viruses.

Rnf1 is the primary electron source to nitrogenase in a high-ammonium-accumulating strain of Azotobacter vinelandii.

The enzyme nitrogenase performs the process of biological nitrogen fixation (BNF), converting atmospheric dinitrogen gas into the biologically accessible ammonia, which is rapidly protonated at physiological pH to yield ammonium. The reduction of dinitrogen requires both ATP and electrons. Azotobacter vinelandii is an aerobic nitrogen-fixing microbe that is a model organism for the study of BNF. Previous reports have described strains of A. vinelandii that are partially deregulated for BNF, resulting in the release of large quantities of ammonium into the growth medium. Determining the source of the electrons required to drive BNF is complicated by the existence of several protein complexes in A. vinelandii that have been linked to BNF in other species. In this work, we used the high-ammonium-accumulating strains of A. vinelandii to probe the source of electrons to nitrogenase by disrupting the Rnf1 and Fix complexes. The results of this work demonstrate the potential of these strains to be used as a tool to investigate the contributions of other enzymes or complexes in the process of BNF. These results provide strong evidence that the Rnf1 complex of A. vinelandii is the primary source of electrons delivered to the nitrogenase enzyme in this partially deregulated strain. The Fix complex under native regulation was unable to provide sufficient electrons to accumulate extracellular ammonium in the absence of the Rnf1 complex. Increased ammonium accumulation could be attained in a strain lacking the Rnf1 complex if the genes of the Fix protein complex were relocated behind the strong promoter of the S-layer protein but still failed to achieve the levels found with just the Rnf1 complex by itself. KEY POINTS: • The Rnf1 complex is integral to ammonium accumulation in A. vinelandii. • The Fix complex can be deleted and still achieve ammonium accumulation in A. vinelandii. • A. vinelandii can be engineered to increase the contribution of the Fix complex to ammonium accumulation.

Fur Skin – A Valuable Material, Considerations on Quality Assessment

This work is intended to reveal the most important physico-chemical and physico-mechanical characteristics of the fur skins that determine their quality. Values of these characteristics are discussed as depending on the semifinished product related to technological significance and consequence for the user. The main characteristics and values for fine furs are proposed, thus providing some guidelines in evaluating their quality. Investigations in this field can lead to the determination of some new quality characteristics of woolen sheepskins and fine furs. A case study on fine and common furskins is presented and reveals the uniqueness and versatility of the combination of two protein layers, represented by natural furs, composed of collagen and keratin, unmatched by any synthetic material.

Dynamic Profile of S-Layer Proteins Controls Surface Properties of Emetic Bacillus cereus AH187 Strain

Many prokaryotes are covered by a two-dimensional array of proteinaceous subunits. This surface layers (S-layer) is incompletely characterized for many microorganisms. Here, we studied Bacillus cereus AH187. A genome analysis identified two genes encoding the S-layer proteins SL2 and EA1, which we experimentally confirmed to encode the two protein components of the S-layer covering the surface of B. cereus. Shotgun proteomics analysis indicated that SL2 is the major component of the B. cereus S-layer at the beginning of exponential growth, whereas EA1 becomes more abundant than SL2 during later stages of stationary growth. Microscopy analysis revealed the spatial organization of SL2 and EA1 at the surface of B. cereus to depend on their temporal-dynamics during growth. Our results also show that a mutant strain lacking functional SL2 and EA1 proteins has distinct surface properties compared to its parental strain, in terms of stiffness and hydrophilicity during the stationary growth phase. Surface properties, self-aggregation capacity, and bacterial adhesion were observed to correlate. We conclude that the dynamics of SL2 and EA1 expression is a key determinant of the surface properties of B. cereus AH187, and that the S-layer could contribute to B. cereus survival in starvation conditions.

Comparative proteomic analyses of resistant and susceptible crucian carp to Gyrodactylus kobayashii infection

Gyrodactylid monogeneans are common parasites of teleost fishes characterized by high host specificity. Host specificity and its underlying mechanisms of monogenean have been always a fascinating issue that has attracted considerable attention from researchers. However, the molecular mechanism responsible for the host specificity of monogenean is still not yet fully understood. In this study, the susceptibility to Gyrodactylus kobayashii of different species or varieties in the genus Carassius was investigated. Furthermore, the proteome changes in the skin mucus of resistant and susceptible fish were analyzed by Tandem Mass Tags (TMT) quantitative proteomics method. The experimental results indicated that G. kobayashii was found in 12 varieties of goldfish and ChangFeng C. auratus gibelio with different degrees of infection percentage, while no G. kobayashii was found in C. auratus gibelio var. CAS Ⅴ. Population dynamics of G. kobayashii on red white common goldfish and C. auratus gibelio var. CAS Ⅴ showed distinct trends in the in vivo infection experiment. These results demonstrated C. auratus gibelio var. CAS Ⅴ is a resistant fish to G. kobayashii. Proteomic analysis revealed that 41 differentially expressed proteins (DEPs), including 25 up-regulated and 16 down-regulated proteins, were identified. Bioinformatics analysis indicated these DEPs were involved in multiple biological functions including immunity, metabolism, and signal transduction. Among them, some immune-related proteins, such as cadherin-1, serotransferrin, and glutathione transferase in the fish mucus might be potential determinants of the host susceptibility to G. kobayashii. Also, some structural and energy metabolism-related DEPs, such as small muscular protein, keratin and vitelline membrane outer layer protein served as a source of nutrition and might also affect host susceptibility. Overall, this study not only provided systematic protein information of crucian carp skin mucus but also identified some potential candidate proteins that might be related to host susceptibility to G. kobayashii.

Differences in Protein Profiles of Kefir Grains from Different Origins When Subcultured in Goat Milk.

Proteins not only serve as a nitrogen source for microorganisms but are the main skeleton of kefir grains. After subculturing in goat milk for 4 months, proteins and peptides in three kefir grains from China, Germany, and the United States were analyzed. Except for the S-layer protein from special Lactobacillus sp., αs1-casein, αs2-casein, and β-casein from goat milk were found in kefir grains. These proteins could form aggregates through a covalent interaction with polysaccharides to maintain the morphological stability of the grains. Furthermore, they were highly related to the microbiota in kefir grains. Additionally, a number of hydrophilic/hydrophobic peptides that were hydrolyzed by extracellular proteases were found from kefir grains. A correlation may exist between peptides and Lactobacillus sp. in kefir grains. Bioactive peptides, including DKIHPF, LGPVRGPFP, and QEPVLGPVRGPFP, were found from these kefir grains. The results indicated that goat milk as a substrate affects the protein and peptide composition of kefir grains.

Effect of enamel-surface modifications on shear bond strength using different adhesive materials

BackgroundThis study aimed to investigate the effect of enamel-surface modifications on the shear bond strength between ceramic brackets bonded using different adhesive materials and the enamel surface and to identify the most suitable clinical adhesive and bonding method. Whether the non-acid-etching treatment met the clinical bond strength was also determined.MethodsA total of 108 extracted premolars were divided into nine groups (n = 12) based on the different enamel-surface modification techniques (acid etching, deproteinization, and wetting). Group 1 was bonded with Transbond™ XT adhesive, whereas groups 2–9 were bonded with resin-modified glass ionomer cement (RMGIC). The treatment methods for each group were as follows: groups 1 and 2, acid etching; group 3, acid etching and wetting; group 4, acid etching and deproteinization; group 5, acid etching, deproteinization, and wetting; group 6, deproteinization; group 7, deproteinization and wetting; group 8, without treatment; and group 9, wetting. The samples' shear bond strength was measured using an universal testing machine. Adhesive remnant index (ARI) was examined using a stereomicroscope. The enamel-surface morphology was observed with a scanning electron microscope. One-way ANOVA with Tukey’s post-hoc test and chi-square test were used for statistical analysis, and p < 0.05 and α = 0.05 were considered statistically significant.ResultsThe ARIs of groups 1–5 and 6–9 were statistically significant (p = 0.000). The enamel surface of groups 1–5 was demineralized, and only a tiny amount of protein remained in groups 7 and 8, whereas a thick layer of protein remained in groups 8 and 9.ConclusionsRMGIC adhesive did not damage the enamel surface and achieved the required clinical bond strength. The enamel surface was better treated with 5.25% sodium hypochlorite preferably under non-acid-etching conditions.

Inactivation of viruses related to foodborne infections using cold plasma technology

AbstractGlobally, there is a rise in day‐to‐day demand for minimally processed foods to supply nutritious, wholesomeness and safe foods to the consumers. Contamination of food by pathogens is a serious problem resulting in several outbreaks. Food pathogens like molds, bacteria were detectable and can be inactivated. The virus detection in foods is always a difficult task as their presence could not alter any noticeable change in the quality. Norovirus, Hepatitis A viruses are well‐known for their foodborne outbreaks and illnesses. Enveloped viruses are resistant and have the stability to the current traditional preservation methods due to the presence of a protective capsid layer and an envelope. The current thermal processing has shown significant effect on the product quality. The use of chemical disinfestation compounds is not suitable for food commodities. There is a need for alternative nonthermal food processing technologies for decontamination of food and food packages and preserving the food quality as well. Cold plasma is one of the emerging nonthermal, chemical‐free residues, and eco‐friendly technology widely being applied to the different food sectors. The main antiviral mechanism is the disruption of the capsid protein layer, the oxidation and denaturation of viral proteins. The method has also caused damage to the envelope layer and genetic material. This review focuses on cold plasma inactivation efficiency on different viruses.

Nanobiotechnological prospects of probiotic microflora: Synthesis, mechanism, and applications

Nanotechnology-driven solutions have almost touched every aspect of life, such as therapeutics, cosmetics, agriculture, and the environment. Physical and chemical methods for the synthesis of nanoparticles involve hazardous reaction conditions and toxic reducing as well as stabilizing agents. Hence, environmentally benign green routes are preferred to synthesize nanoparticles with tunable size and shape. Bacteria, fungi, algae, and medicinal plants are employed to synthesize gold, silver, copper, zinc, and other nanoparticles. However, very little literature is available on exploring probiotic bacteria for the synthesis of nanoparticles. In view of the background, this review gives the most comprehensive report on the nanobiotechnological potential of probiotic bacteria like Bacillus licheniformis, Bifidobacterium animalis, Brevibacterium linens, Lactobacillus acidophilus, Lactobacillus casei, and others for the synthesis of gold (AuNPs), selenium (SeNPs), silver (AgNPs), platinum (PtNPs), tellurium nanoparticles (TeNPs), zinc oxide (ZnONPs), copper oxide (CuONPs), iron oxide (Fe3O4NPs), and titanium oxide nanoparticles (TiO2NPs). Both intracellular and extracellular synthesis are involved as potential routes for biofabrication of polydispersed nanoparticles that are spherical, rod, or hexagonal in shape. Capsular exopolysaccharide associated carbohydrates such as galactose, glucose, mannose, and rhamnose, cell membrane-associated diglycosyldiacylglycerol (DGDG), 1,2-di-O-acyl-3-O-[O-α-D-galactopyranosyl-(1 → 2)-α-d-glucopyranosyl]glycerol, triglycosyl diacylglycerol (TGDG), NADH-dependent enzymes, amino acids such as cysteine, tyrosine, and tryptophan, S-layer proteins (SLP), lacto-N-triose, and lactic acid play a significant role in synthesis and stabilization of the nanoparticles. The biogenic nanoparticles can be recovered by rational treatment with sodium dodecyl sulfate (SDS) and/or sodium hydroxide (NaOH). Eventually, diverse applications like antibacterial, antifungal, anticancer, antioxidant, and other associated activities of the bacteriogenic nanoparticles are also elaborated. Being more biocompatible and effective, probiotic-generated nanoparticles can be explored as novel nutraceuticals for their ability to ensure sustained release and bioavailability of the loaded bioactive ingredients for diagnosis, targeted drug delivery, and therapy.

The effects of thermal treatment on emulsifying properties of soy protein isolates: Interfacial rheology and quantitative proteomic analysis

In this work, the interfacial rheological properties and the quantitative changes of proteins at interfacial protein layers of emulsions stabilized by soy protein isolates (SPI) and heat-treated soy protein isolates (HSPI) were investigated. The quantification results showed that the relative quantities of albumin (2S) and glycinin (11S) in SPI decreased at the oil-water interface, suggesting that they possessed lower interfacial affinities at the interface. Basic 7S globulin presented more adsorption at the oil-water interface due to the well balance of the hydrophobic and hydrophilic groups of its amino acid sequence. The HSPI (95 °C, 20 min) showed a larger apparent diffusion rate (Kdiff) and a shorter equilibrium adsorption time. The results of interfacial rheology of globulins were consistent with their interfacial quantitative changes, which demonstrated that the interfacial behavior and adsorption ability of globulin were improved by thermal treatment. In this research, the interfacial behaviors of SPI and HSPI was illustrated by their interfacial properties and quantitative results of interfacial adsorbed protein layers, would promote a profound comprehension for the interfacial behavior of the protein and the influence of thermal treatment on protein interfacial properties.

Design and Fabrication of a Dual Protein-Based Trilayered Nanofibrous Scaffold for Efficient Wound Healing.

Chronic wound healing is a major threat all over the world. There are currently a plethora of biomaterials-based wound dressings available for wound healing applications. In this study, a dual protein-based (silk fibroin and sericin) nanofibrous scaffold from a natural source (B.mori silkworm cocoons) with antibacterial and antioxidative properties for wound healing was investigated. An electrospun layer-by-layer silk protein-based nanofibrous scaffold was fabricated with a top layer of hydrophobic silk fibroin protein blended with polyvinyl alcohol (PVA), a middle layer of waste protein silk sericin loaded with silver(I) sulfadiazine as an antibacterial agent, and a bottom layer using silk fibroin blended with polycaprolactone (PCL). The trilayered nanofibrous scaffold with a smooth and bead-free morphology demonstrated excellent wettability, slow in vitro degradation, controlled drug release, and potent antibacterial and antioxidant properties. In vitro, the scaffold also demonstrated excellent hemocompatibility and biocompatibility. Furthermore, in vivo wound contraction, histological, and micro-CT investigations show complete wound healing and the formation of new skin tissue in a male Balb/c mouse model treated with the scaffold. The antioxidant properties of the sericin protein and SSD-based triple-layered nanofibrous scaffold protect the wound from bacterial infection and improve wound healing in a mouse model. The current study develops a dual protein-based nanofibrous scaffold with antibacterial and antioxidant properties as a promising wound dressing material.

Adsorption of lysozyme and antibodies at material surfaces: Implications to material compatibility for development of biologics

The impacts of contact materials such as borosilicate glass, polycarbonate (PC) and cellulose, on the adsorption of lysozyme, along with the interaction of an immunoglobulin (IgG) with polyethersulfone (PES)-typical filter material, were evaluated using quartz crystal microbalance with dissipation (QCM-D). The adsorption and viscoelastic properties of the adsorbed protein layers, along with displacement and adsorption prevention by polysorbate 80 (PS80) and poloxamer 188, were characterized based on frequency and dissipation change. A decrease in frequency denotes a mass gain from adsorption, while an increase in dissipation indicates formation of a viscoelastic layer. The adsorption at a fixed lysozyme concentration increases in the following order: borosilicate glass > PC > cellulose. Furthermore, the adsorbed protein layers on PC surface appears to be the most viscoelastic, whereas the adsorbed layer at borosilicate seems to be the least viscoelastic. Finally, adsorption at interfaces is irreversible and cannot be completely displaced by PS80 or poloxamer 188. However, the adsorption of IgG on PES was shown to be effectively prevented when PS80 is present in the formulation. These results provide insights in understanding material compatibilities during manufacturing, storage and administration of therapeutic proteins in biopharmaceutical industry.

Advanced characterization of albumin adsorption on a chemically treated surface for osseointegration: An innovative experimental approach

Surface chemistry, charge, wettability, and roughness affect the adsorbed protein layer, influencing biocompatibility and functionality of implants. Material engineering seeks innovative, sensitive, and reliable characterization techniques for study the adsorbed proteins. These techniques must be suitable to be directly used on the surfaces of clinical interest. In this paper, the characterization of surfaces with topography and chemistry developed for osseointegration is performed by innovative surface analysis techniques to investigate the properties of adsorbed bovine serum albumin. Ti6Al4V alloy chemically treated with an oxidative process to obtain peculiar surface features (roughness and surface hydroxylation) was tested and compared with mirror-polished titanium. Albumin forms a continuous layer on both Ti surfaces when adsorbed from near physiological concentrations, as proved by Kelvin force probe microscopy. It was observed that the hydroxylation degree plays a pivotal role in determining the conformation of proteins after adsorption, where it strongly drives protein unfolding, as confirmed by Surface Enhanced Raman scattering, and in influencing the mechanism and chemical stability of protein-surface interactions, which was highlighted by zeta potential titration curves.