Fructosylation of human insulin causes AGEs formation, structural perturbations and morphological changes: an in silico and multispectroscopic study.

Fructosylation of proteins results in the formation of advanced glycation end-products (AGEs). A diet rich in fructose along with hyperglycemia can cause fructose mediated glycation (fructosylation) of proteins, which results in AGEs formation. Insulin is a peptide hormone that is glycated when exposed to carbohydrates such as glucose. In this study, we have analysed the interaction of insulin with fructose and biophysically characterized fructose modified insulin. In silico studies performed through molecular docking and molecular dynamics simulation revealed that fructose binds to insulin with strong affinity resulting in the formation of insulin-fructose complex. Fructosylation of insulin caused hyperchromicity, loss of intrinsic fluorescence, gain in AGEs specific fluorescence and elevated the carbonyl and fructosamine content. Enhanced thioflavin T fluorescence suggested the presence of fibrillar structures at higher concentrations of fructose. Electron microscopy revealed the formation of characteristic amorphous and amyloid like aggregates at lower and higher concentrations of fructose, respectively. These findings show that fructosylation of insulin causes AGEs production, aggregation and alters its gross structural integrity. These changes may reduce the biological activity of insulin that can aggravate conditions like type II diabetes mellitus.Communicated by Ramaswamy H. Sarma.

4-Chloro-1,2-phenylenediamine induced structural perturbation and genotoxic aggregation in human serum albumin.

4-Chloro-1,2-phenylenediamine (4-Cl-OPD) is a halogenated aromatic diamine used as a precursor in permanent hair color production. Despite its well-documented mutagenic and carcinogenic effects in various in vitro and in vivo models, its role in fibrillar aggregate formation and their genotoxic effect in therapeutic proteins has received less attention. The significance of human serum albumin (HSA) arises from its involvement in bio-regulatory and transport processes. HSA misfolding and aggregation are responsible for some of the most frequent neurodegenerative disorders. We used various complementary approaches to track the formation of amyloid fibrils and their genotoxic effect. Molecular dynamics study demonstrated the complex stability. The impact of 4-Cl-OPD on the structural dynamics of HSA was confirmed by Raman spectroscopy, X-ray diffraction, HPLC and SDS-PAGE. Fibrilllar aggregates were investigated using Congo red assay, DLS, and SEM. The genotoxic nature of 4-Cl-OPD was confirmed using plasmid nicking assay and DAPI staining, which revealed DNA damage and cell apoptosis. 4-Cl-OPD provides a model system for studying fibrillar aggregation and their genotoxic potential in the current investigation. Future studies should investigate the inhibition of the aggregation/fibrillation process, which may yield valuable clinical insights.

Effect of phosphonate functional group on corrosion inhibition of imidazoline derivatives in acidic environment.

In the present study, ({2-[2-(7-Isopropyl-1,4-dimethyl-9,10-octahydro-phenanthren-1-yl)-4,5-dihydro-1-yl]-ethylamino}-methyl)-phosphonicimidazole (GSIM) was synthesized by introducing phosphonate (-PH2O3) group into imidazoline derivatives and its corrosion inhibition performance was studied for Q345 steel in acidic medium along with SIM using potentiodynamic polarization, EIS and SECM analysis. Surface analysis of steel samples was also performed by optical microscopy and SEM-EDS analysis after polarization tests. XPS was used to detect chemical composition of the surface passive films. It was observed that introducing -PH2O3 group not only improved the adsorption capacity on the metal surface by coordinating with iron ions, but also inhibited the interference of hydrogen bond formed by -NH2 group and water molecules on the adsorption. GSIM not only inhibited intercrystalline corrosion and pitting corrosion, but also reduced uniform corrosion. Thermodynamics studies demonstrated that GSIM followed the Langmuir adsorption isotherm and had a larger adsorption equilibrium constant than SIM, which indicated that it had a stronger adsorption capacity. XPS and UV confirmed that the coordination between GSIM and Fe3+ and hydrogen bonding between SIM and water molecules. The quantum chemical study further clarified that the site and strength of hydrogen bond between SIM and H2O and the dominant configuration and coordination stability of GSIM with Fe3+.

Hydroxyl radical induced structural perturbations make insulin highly immunogenic and generate an auto-immune response in type 2 diabetes mellitus.

Reactive oxygen species (ROS) cause oxidative damage to proteins and generate deleterious by-products which induce a breakdown of immune tolerance and produce antibodies against host macromolecules with implication in human diseases. This study characterizes the hydroxyl radical (OH) modifications of insulin, evaluates its cytotoxicity and immunogenicity, and probes its role in type 2 diabetes (T2DM) autoimmunity. The results demonstrate susceptibility of insulin to modifications induced by OH, causing exposure of its chromophoric aromatic amino acid residues, quenching of tyrosine fluorescence intensity, loss of α-helix and gain in ß content. Modification causes re-arrangement of native interactions of the aromatic residues in insulin. It enhanced the carbonyl content in insulin, exposed its hydrophobic patches and generated non-fibrillar, amorphous type of aggregates that are cytotoxic in nature. Native insulin induced low titre antibodies in immunized rabbits, whereas OH modified insulin generated a strong immune response. Competitive ELISA studies showed high specificity of antibodies generated against OH modified insulin towards the modified protein. Cross reaction studies showed the presence of common antigenic determinants on various oxidised proteins. Since T2DM patients show increased ROS production, oxidation of insulin is expected to occur, which might amplify autoimmune reactions against insulin. True to the assumption, direct binding ELISA showed the presence of anti-OH insulin circulating antibodies in T2DM patients which are specific for the oxidized insulin. In conclusion, insulin loses structural integrity to OH, forms cytotoxic amorphous aggregates, turns highly immunogenic and elicits humoral response in T2DM patients.

Corrosion performance of various deformed surfaces of implant steel for coronary stent applications: Effect of protein concentration.

This work was done to systematically elucidate the corrosion behavior of austenitic stainless steel subjected to various degree of cold deformation (10 %, 20 % & 30 %). The experiments were performed in phosphate buffer saline (PBS) solution having different concentrations of bovine serum albumin (0.2, 0.5, 1.0, 2.0, 4.0 g L-1). Potentiodynamic polarization tests and electrochemical impedance spectroscopy (EIS) analysis were performed to obtain the corrosion parameters. Scanning electron microscopy with energy dispersive X-ray (SEM-EDX), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to determine the surface morphologies and chemical compositions of the surface films. Contact angle analysis was also used to detect the hydrophilic character of sample surfaces. The BSA had a considerable effect of inhibition on the corrosion of SSs in annealed as well as in deformed state due to its adsorption on surface of steel. For annealed samples, at 4.0 g L-1BSA concentration, the corrosion resistance was drastically decreased but interestingly not for sample with more than 10 % deformation and the concentration effect of BSA is also not very significant after 0.5 g L-1 for deformed surfaces. The breakdown potential for 30 % deformed sample is quite higher in presence of BSA even at 4.0 g L-1 while it is lowest for annealed samples in the same condition. The variation in contact angle with deformation is very less after adsorption of BSA. On the basis of the obtained results, mechanism aspect for corrosion of steel in presence of protein is also deliberated.

Imidazolium-based Ionic Liquid as Efficient Corrosion Inhibitor for AA 6061 Alloy in HCl Solution.

The corrosion inhibition performance of an imidazolium-based ionic liquid (IL), 1-butyl-3-methylimidazolium thiocyanate (BMIm), was studied on AA 6061 alloy in 1 M HCl solution at 303 K, 333 K, and 363 K by gravimetric tests, potentiodynamic polarization, and electrochemical impedance spectroscopy (EIS) analysis. Scanning electron microscopy with energy dispersive X-ray (SEM-EDX) and X-ray photoelectron spectroscopy (XPS) were used to detect the surface morphologies and chemical composition of the surface films. The results indicate that this IL inhibits AA 6061 corrosion in acid with maximum inhibition efficiencies of 98.2%, 86.6%, and 41.2% obtained at 303 K, 333 K, and 363 K respectively. Inhibition efficiency generally decreased with increasing immersion time; the major exception was at 303 K, whereby the inhibition efficiency was detected to increase with immersion time from 30 to 90 min and then decrease slightly beyond 90 min. The results indicate that BMIm is a mixed-type inhibitor with a predominant effect on cathodic reactions. Surface morphology analyses by SEM revealed less surface damage in the presence of the inhibitor. XPS analysis established the development of a protective film on the AA 6061 surface which was hydrophobic in nature.

Role of protein adsorption in the bio corrosion of metallic implants - A review.

Implants are exposed to a complex physiological environment that contains various organic compounds, especially proteins. The adsorption of proteins has an immense influence on the corrosion, biocompatibility and wear properties of implantable metals. Proteins engage in multiple processes that could potentially inhibit or promote metal degradation, depending on the type of proteins, their concentration and the properties of the implant material. In the bio corrosion process, proteins are denatured and transform into a film on the metal surface, inhibiting corrosion. This film is found on many retrieved artificial joints, especially on worn areas, and can protect the passive film from scrapping due to its lubricating effect, thus decreasing tribocorroion. On the other hand, the interactions of metal ions with proteins (and amino acids) create colloidal organometallic complexes. Transport of the complex compounds away from the interface increases dissolution rates; thus, it accelerates the corrosion of metallic implants. The influence of protein adsorption on the corrosion behaviour of metallic biomaterials is presented in this review. Biocompatible metals that are favourably used as implants such as stainless steel, Co-Cr alloys, Ti alloys and biodegradable Mg and Fe alloys are specifically addressed. We have highlighted the adsorption phenomenon of protein on metallic implants, the interaction of proteins with metallic implants and the role of protein adsorption on implant biocorrosion behaviour as well as their wear resistance.

Methylglyoxal modified IgG generates autoimmune response in rheumatoid arthritis.

The detection of autoantibodies generated against modified proteins that stimulate cellular and humoral immune response has developed a lot of interest in the recent years and a search for biomarkers for the early detection of diseases has increased. IgG protein has earned attention for its possible modifications under hyperglycaemic conditions in rheumatoid arthritis, wherein dicarbonyl stress has been reported to alter the structural integrity of the protein. This report suggests that the interaction of the methylglyoxal with the IgG has consequences in the autoimmunopathology of rheumatoid arthritis. Our molecular docking analysis of methylglyoxal and IgG revealed a close interaction between the two molecules. TNBS studies confirmed the interaction by showing a decline in free lysine-arginine content post-MG modifications in IgG. The modified IgG was thermally more stable and showed the generation of glycation adducts N-epsilon-carboxyethyllysine. Rheumatoid arthritis patients showed enhanced carbonyl stress which was expected to induce structural changes in the epitope makeup of IgG. The ELISA studies and gel retardation assay confirmed auto-antibodies against MG modified IgG (MG-IgG) pointing towards the generation of neoepitopes upon IgG after interaction with MG. This study establishes the IgG modification in RA patients under alter carbonyl concentrations.

Heterocyclic Corrosion Inhibitors for J55 Steel in a Sweet Corrosive Medium.

The inhibitory effect of two heterocyclic porphyrin compounds, specifically 5,10,15,20-tetrakis(pentafluorophenyl)-21H,23H-porphyrin palladium(II) (PF-1) and 4,4',4″,4‴-(porphyrin-5,10,15,20-tetrayl)tetrakis(benzoic acid) (PF-2), was studied in a sweet corrosion environment (3.5 wt % NaCl + CO2) on J55 steel by means of weight loss and electrochemical methods. Surface changes were studied using contact angle, scanning electrochemical microscopy (SECM), and atomic force microscopy (AFM) techniques. It was established that PF-2 showed the superlative inhibition efficiency of average of about 93% at 400 ppm concentration. The inductive behavior of the J55 steel surface in the presence of inhibitors was confirmed by SECM. The AFM further confirmed that the surface roughness was considerably decreased in the presence of porphyrins. The surface wettability of the steel was also investigated, and the results established the formation of a water-repellant layer on the surface when porphyrins are absorbed and layer became more hydrophobic with PF-2. Thermodynamics studies showed that the inhibition efficiencies of two compounds evaluated by all measurements follow the Langmuir adsorption isotherm.

Effect of nitrogen and cold working on structural and mechanical behavior of Ni-free nitrogen containing austenitic stainless steels for biomedical applications.

This investigation deals with the evaluation of structural and mechanical behavior of deformed (10% and 20% cold work) and annealed (at 1050°C for 15 min followed by water quenching) Ni-free high nitrogen austenitic stainless steels (HNSs). The microstructure was observed by optical micrograph and the mechanical properties were determined by macrohardness and tensile tests. Both stress strain behavior and work hardening behavior were evaluated. HNSs have smaller grain size as compared to low nitrogen steels and no formation of martensite was observed after 20% cold working. Further, it was found that hardness; yield strength and ultimate tensile strength of the steels linearly increases and elongation decreased with nitrogen content and degree of cold working. The strength coefficient was observed to be higher for the high nitrogen steels; it decreased to some extent with degree of cold working. The work hardening exponent was also observed to decrease with degree of cold working. Influence of nitrogen on mechanical properties was mainly related to its effect on solid solution strengthening. X-ray diffraction analysis of annealed as well as deformed alloys further confirmed no evidence for formation of martensite or any other secondary phases. SEM fractography of the annealed and deformed samples after tensile tests indicates predominantly ductile fracture in all specimens.

In-vitro long term and electrochemical corrosion resistance of cold deformed nitrogen containing austenitic stainless steels in simulated body fluid.

This work was focused on the evaluation of the corrosion behavior of deformed (10% and 20% cold work) and annealed (at 1050 °C for 15 min followed by water quenching) Ni-free high nitrogen austenitic stainless steels (HNSs) in simulated body fluid at 37°C using weight loss method (long term), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization. Scanning electron microscopy (SEM) was used to understand the surface morphology of the alloys after polarization test. It has been observed that cold working had a significant influence on the corrosion resistant properties of these alloys. The weight loss and corrosion rates were observed to decrease with increasing degree of cold working and nitrogen content in the alloy. The corrosion resistance of the material is directly related to the resistance of the passive oxide film formed on its surface which was enhanced with cold working and nitrogen content. It was also observed that corrosion current densities were decreased and corrosion potentials were shifted to more positive values. By seeing pit morphology under SEM, shallower and smaller pits were associated with HNSs and cold worked samples, indicating that corrosion resistance increases with increasing nitrogen content and degree of cold deformation. X-ray diffraction profiles of annealed as well as deformed alloys were revealed and there is no evidence for formation of martensite or any other secondary phases.

Effect of cold working on biocompatibility of Ni-free high nitrogen austenitic stainless steels using Dalton's Lymphoma cell line.

The aims of the present work are to explore the effect of cold working on in-vitro biocompatibility of indigenized low cost Ni-free nitrogen containing austenitic stainless steels (HNSs) and to compare it with conventionally used biomedical grade, i.e. AISI 316L and 316LVM, using Dalton's Lymphoma (DL) cell line. The MTT assay [3-(4,5-dimethythiazol 2-yl)-2,5-diphenyltetrazolium bromide] was performed on DL cell line for cytotoxicity evaluation and cell adhesion test. As a result, it was observed that the HNS had higher cell proliferation and cell growth and it increases by increasing nitrogen content and degree of cold working. The surface wettability of the alloys was also investigated by water contact angle measurements. The value of contact angles was found to decrease with increase in nitrogen content and degree of cold working. This indicates that the hydrophilic character increases with increasing nitrogen content and degree of cold working which further attributed to enhance the surface free energy (SFE) which would be conducive to cell adhesion which in turn increases the cell proliferation.

A review on nickel-free nitrogen containing austenitic stainless steels for biomedical applications.

The field of biomaterials has become a vital area, as these materials can enhance the quality and longevity of human life. Metallic materials are often used as biomaterials to replace structural components of the human body. Stainless steels, cobalt-chromium alloys, commercially pure titanium and its alloys are typical metallic biomaterials that are being used for implant devices. Stainless steels have been widely used as biomaterials because of their very low cost as compared to other metallic materials, good mechanical and corrosion resistant properties and adequate biocompatibility. However, the adverse effects of nickel ions being released into the human body have promoted the development of "nickel-free nitrogen containing austenitic stainless steels" for medical applications. Nitrogen not only replaces nickel for austenitic structure stability but also much improves steel properties. Here we review the harmful effects associated with nickel and emphatically the advantages of nitrogen in stainless steel, as well as the development of nickel-free nitrogen containing stainless steels for medical applications. By combining the benefits of stable austenitic structure, high strength, better corrosion and wear resistance and superior biocompatibility in comparison to the currently used austenitic stainless steel (e.g. 316L), the newly developed nickel-free high nitrogen austenitic stainless steel is a reliable substitute for the conventionally used medical stainless steels.