Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region.

The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

Exploring the significance of potassium homeostasis in copper ion binding to human αB-Crystallin.

αB-Crystallin (αB-Cry) is a small heat shock protein known for its protective role, with an adaptable structure that responds to environmental changes through oligomeric dynamics. Cu(II) ions are crucial for cellular processes but excessive amounts are linked to diseases like cataracts and neurodegeneration. This study investigated how optimal and detrimental Cu(II) concentrations affect αB-Cry oligomers and their chaperone activity, within the potassium-regulated ionic-strength environment. Techniques including isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, inductively coupled plasma atomic emission spectroscopy, cyclic voltammetry, dynamic light scattering, circular dichroism, and MTT assay were employed and complemented by computational methods. Results showed that potassium ions affected αB-Cry's structure, promoting Cu(II) binding at multiple sites and scavenging ability, and inhibiting ion redox reactions. Low concentrations of Cu(II), through modifications of oligomeric interfaces, induce regulation of surface charge and hydrophobicity, resulting in an increase in chaperone activity. Subunit dynamics were regulated, maintaining stable interfaces, thereby inhibiting further aggregation and allowing the functional reversion to oligomers after stress. High Cu(II) disrupted charge/hydrophobicity balance, sewing sizable oligomers together through subunit-subunit interactions, suppressing oligomer dissociation, and reducing chaperone efficiency. This study offers insights into how Cu(II) and potassium ions influence αB-Cry, advancing our understanding of Cu(II)-related diseases.

Development and evaluation of anti-reflux functional-oral suspension raft composed of sodium alginate-mung bean protein complex.

This study aimed to develop a sodium alginate (Na alginate) and mung bean protein (MBP) raft complex to improve gastric reflux symptoms. Na alginate and MBP complexes with different ratios (1:1, 2:1, and 3:1, respectively) were used for raft formulations through a wet Maillard reaction. Structural properties of raft strength, reflux resistance, intrinsic fluorescence emission spectroscopy, and Fourier transform infrared spectroscopy (FTIR) were investigated for rafts. The suspension 1:1 Na alginate/MBP with 0 h Maillard reaction time exhibited the lowest sedimentation volume among the suspensions. In contrast, 3:1 Na alginate/MBP with 6 h Maillard reaction time showed the highest sedimentation volume. Based on the results, the 3:1 Na alginate/MBP rafts had the best results, and the results were within acceptable limits. Functional properties, including antioxidant properties, the Helicobacter pylori inhibition assay, the pancreatic lipase inhibition assay, and angiotensin-converting enzyme (ACE) inhibition, were investigated for rafts. The Na alginate/MBP raft has similar characteristics to Gaviscon syrup and can be used for obesity, Helicobacter pylori infection, high blood pressure, and gastric reflux.

Chemical Modification of the Amino Groups of Human Insulin: Investigating Structural Properties and Amorphous Aggregation of Acetylated Species.

The efficacy of human recombinant insulin can be affected by its aggregation. Effects of acetylation were observed on insulin structure, stability, and aggregation at 37 and 50 °C and pH of 5.0 and 7.4 with the use of spectroscopy, circular dichroism (CD), dynamic light scattering (DLS), and atomic force microscopy (AFM). Raman and FTIR results were indicative of structural changes in AC-INS, and CD analyses showed a slight increase in ß-sheet content in AC-INS. Melting temperature (Tm) measurements indicated an overall more stable structure and spectroscopic assessment showed a more compact one. Formation of amorphous aggregates was followed over time and kinetics parameters showed a longer nucleation phase (higher t* amount) and lower aggregates amount (lower Alim) for acetylated insulin (AC-INS) compared to native (N-INS) in all tested conditions. The results of amyloid-specific probes approved the formation of amorphous aggregates. Size particle and microscopic analysis suggested that AC-INS was less prone to form aggregates, which were smaller if formed. In conclusion, this study has demonstrated that controlled acetylation of insulin may lead to its higher stability and lower propensity toward amorphous aggregation and has provided insight into the result of this type of post-translational protein modification.

Design, optimization and characterization of a novel antibacterial chitosan-based hydrogel dressing for promoting blood coagulation and full-thickness wound healing: A biochemical and biophysical study.

The use of hydrogel dressings has become increasingly popular as a scaffold for skin tissue engineering. Herein, we have developed an innovative wound dressing using chitosan, fibrinogen, nisin, and EDTA as an effective antibacterial scaffold for wound treatment. The structural and functional characteristics of the hydrogel, including morphology, mechanical strength, drug encapsulation and release, swelling behaviors, blood coagulation, cytotoxicity, and antibacterial activity, were studied. Spectroscopic studies indicated that the attachment of chitosan to fibrinogen is associated with minimal change in its secondary structure; subsequently, at higher temperatures, it is expected to preserve fibrinogen's conformational stability. Mechanical and blood coagulation analyses indicated that the incorporation of fibrinogen into the hydrogel resulted in accelerated clotting and enhanced mechanical properties. Our cell studies showed biocompatibility and non-toxicity of the hydrogel along with the promotion of cell migration. In addition, the prepared hydrogel indicated an antibacterial behavior against both Gram-positive and Gram-negative bacteria. Interestingly, the in vivo data revealed enhanced tissue regeneration and recovery within 17 days in the studied animals. Taken together, the results obtained from in vitro and histological assessments indicate that this innovatively designed hydrogel shows good potential as a candidate for wound healing.

Neuroprotective Effect of Propolis Polyphenol-Based Nanosheets in Cellular and Animal Models of Rotenone-Induced Parkinson's Disease.

Considering the central role of oxidative stress in the onset and progress of Parkinson's diseases (PD), search for compounds with antioxidant properties has attracted a growing body of attention. Here, we compare the neuroprotective effect of bulk and nano forms of the polyphenolic fraction of propolis (PFP) against rotenone-induced cellular and animal models of PD. Mass spectrometric analysis of PFP confirmed the presence of multiple polyphenols including kaempferol, naringenin, coumaric acid, vanillic acid, and ferulic acid. In vitro cellular experiments indicate the improved efficiency of the nano form, compared to the bulk form, of PFP in attenuating rotenone-induced cytotoxicity characterized by a decrease in cell viability, release of lactate dehydrogenase, increased ROS generation, depolarization of the mitochondrial membrane, decreased antioxidant enzyme activity, and apoptosis induction. In vivo experiments revealed that while no significant neuroprotection was observed relating to the bulk form, PFP nanosheets were very effective in protecting animals, as evidenced by the improved behavioral and neurochemical parameters, including decreased lipid peroxidation, increased GSH content, and antioxidant enzyme activity enhancement. We suggest that improved neuroprotective effects of PFP nanosheets may be attributed to their increased water solubility and enrichment with oxygen-containing functional groups (such as OH and COOH), leading to increased antioxidant activity of these compounds.

The anti-platelet drug ticlopidine inhibits FapC fibrillation and biofilm production: Highlighting its antibiotic activity.

Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.

Bivalent metal ions induce formation of α-synuclein fibril polymorphs with different cytotoxicities.

α-Synuclein (α-Syn) aggregates are key components of intracellular inclusion bodies characteristic of Parkinson's disease (PD) and other synucleinopathies. Metal ions have been considered as the important etiological factors in PD since their interactions with α-Syn alter the kinetics of fibrillation. In the present study, we have systematically explored the effects of Zn2+, Cu2+, Ca2+, and Mg2+ cations on α-Syn fibril formation. Specifically, we determined fibrillation kinetics, size, morphology, and secondary structure of the fibrils and their cytotoxic activity. While all cations accelerate fibrillation, we observed distinct effects of the different ions. For example, Zn2+ induced fibrillation by lower tlag and higher kapp and formation of shorter fibrils, while Ca2+ ions lead to formation of longer fibrils, as evidenced by dynamic light scattering and atomic force microscopy studies. Additionally, the morphology of formed fibrils was different. Circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopies revealed higher contents of ß-sheets in fibrils. Interestingly, cell viability studies indicated nontoxicity of α-Syn fibrils formed in the presence of Zn2+ ions, while the fibrils formed in the presence of Cu2+, Ca2+, and Mg2+ were cytotoxic. Our results revealed that α-Syn fibrils formed in the presence of different divalent cations have distinct structural and cytotoxic features.

Designing a new alginate-fibrinogen biomaterial composite hydrogel for wound healing.

Wound healing is a complex process and rapid healing necessitates a proper micro-environment. Therefore, design and fabrication of an efficacious wound dressing is an impressive innovation in the field of wound healing. The fabricated wound dressing in this scenario was designed using a combination of the appropriate coagulating and anti-bacterial materials like fibrinogen (as coagulating agent), nisin (as anti-bacterial agent), ethylenediaminetetraacetic acid (as anti-bacterial agent), and alginate (as wound healing agent). Biophysical characterization showed that the interaction of fibrinogen and alginate was associated with minor changes in the secondary structure of the protein. Conformational studies showed that the protein was structurally stable at 42 °C, is the maximum temperature of the infected wound. The properties of the hydrogel such as swelling, mechanical resistance, nisin release, antibacterial activity, cytotoxicity, gel porosity, and blood coagulation were assessed. The results showed a slow release for the nisin during 48 h. Antibacterial studies showed an inhibitory effect on the growth of Gram-negative and Gram-positive bacteria. The hydrogel was also capable to absorb a considerable amount of water and provide oxygenation as well as incorporation of the drug into its structure due to its sufficient porosity. Scanning electron microscopy showed pore sizes of about 14-198 µm in the hydrogel. Cell viability studies indicated high biocompatibility of the hydrogel. Blood coagulation test also confirmed the effectiveness of the synthesized hydrogel in accelerating the process of blood clot formation. In vivo studies showed higher rates of wound healing, re-epithelialization, and collagen deposition. According to the findings from in vitro as well as in vivo studies, the designed hydrogel can be considered as a novel attractive wound dressing after further prerequisite assessments.

A dual responsive robust human serum albumin-based nanocarrier for doxorubicin.

Targeted drug therapy against cancer has been introduced as a smart strategy to combat the unwanted side effects due to systemic administration of chemotherapeutics. A human serum albumin (HSA)-based nanocarrier was fabricated with the aim to target reductive media and acidic pH of the tumor tissues. α-Lipoic acid (LA) was applied to increase the number of disulfide bonds in the nanocarrier to target higher glutathione concentrations present in tumor tissues and polyethylene glycol was used to target the acidic pH of tumors. UV illumination, ethanol desolvation, oxygen bubbling, and a mixture of redox buffers were employed to prepare doxorubicin-loaded HSA-LA nanoparticles. The nanocarrier was supposed to release the loaded doxorubicin in reductive and acidic pH media. Fourier-transform infrared spectroscopy and energy dispersive X-ray analysis indicated successful attachment of LA to HSA. The prepared nanoplatform presented improved doxorubicin loading efficiency and content and successfully released the loaded doxorubicin in the expected conditions. Protein corona study indicated that positively charged plasma proteins with molecular weights of nearly 80 kDa are absorbed to the surface of the nanoparticles. Furthermore, it showed desirable UV and storage stability, which implied its robustness and improved shelf life if applied in nanomedicine.

Role of surface oxygen-containing functional groups of graphene oxide quantum dots on amyloid fibrillation of two model proteins.

There are many reports demonstrating that various derivatives of carbon nanoparticles are effective inhibitors of protein aggregation. As surface structural features of nanoparticles play a key role on modulating amyloid fibrillation process, in the present in vitro study, bovine insulin and hen egg white lysozyme (HEWL) were selected as two model proteins to investigate the reducing effect of graphene oxide quantum dots (GOQDs) on their assembly under amyloidogenic conditions. GOQDs were prepared through direct pyrolysis of citric acid, and the reduction step was carried out using ascorbic acid. The prepared nanoparticles were characterized by UV-Vis, X-ray photoelectron, and FT-IR spectroscopies, transmission electron and atomic force microscopies, zeta potential measurement, and Nile red fluorescence assay. They showed the tendencies to modulate the assembly of the proteins through different mechanisms. While GOQDs appeared to have the capacity to inhibit fibrillation, the presence of reduced GOQDs (rGOQDs) was found to promote protein assembly via shortening the nucleation phase, as suggested by ThT fluorescence data. Moreover, the structures produced in the presence of GOQDs or rGOQDs were totally nontoxic. We suggest that surface properties of these particles may be part of the differences in their mechanism(s) of action.

Biodegradation of phenol and dyes with horseradish peroxidase covalently immobilized on functionalized RGO-SiO2 nanocomposite.

Horseradish peroxidase (HRP) was immobilized onto a functionalized reduced graphene oxide-SiO2 through the covalent bonding process. By using scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR), the formed nanocomposites were characterized. The kinetic parameters including the catalytic constant, kcat, and the catalytic efficiency, kcat/Km, increased 5.5 and 6 times, respectively, after immobilization. The circular dichroism analysis demonstrated that the α-helical content increased from 39% to 46% after immobilization. The immobilization improved the reusability of HRP as 70% of initial activity retained after 10 cycles. Due to the buffering effect, the immobilized HRP was less sensitive to pH changes as compared to the free HRP. At temperature 40 °C and during 90 min, the immobilized HRP retained 90% of the initial activity while 70% of initial activity remained for the free HRP. After 35-day storage, no reduction in the activity was observed for the immobilized HRP. The removal efficiency for phenol concentration (2500 mg/L) obtained 100% and 50% for the immobilized and free HRP, respectively. The results showed that the immobilized HRP promoted the dyes decolorization from 2-fold until 26-fold as compared to the free HRP. The decolorization efficiencies reached 100% for most dyes in the case of immobilized HRP.

Three Pt-Pt Complexes with Donor-acceptor Feature: Anticancer Activity, DNA Binding Studies and Molecular Docking Simulation.

BACKGROUND: Due to their unique properties and potential applications in variety of areas, recently, a special attention is given to the binuclear platinum (II) complexes. They reveal a highly tunable features upon the modification of their cyclometallating and bridging ligands. OBJECTIVE: The aim of this study was to evaluate the anticancer activity and DNA binding affinity of three binuclear platinum (II) complexes, including ht-[(p-FC6H4)Pt(µ-PN)(µ-NP)PtMe2](CF3CO2)(1), ht-[(p- MeC6H4)Pt(µ-PN)(µ-NP)Pt(p MeC6H4) Me] (CF3CO2)(2) and ht-[Pt2Me3(µ-PN)2](CF3CO2) (3). METHODS: MTT assay was performed to study the cell viability of Jurkat and MCF-7 lines against synthesized complexes, followed by apoptosis detection experiments. Several spectroscopic methods with molecular docking simulation were also used to investigate the detail of interaction of these platinum complexes with DNA. RESULTS: Cell viability assay demonstrated a notable level of cytotoxicity for the synthetic platinum complexes. Further studies proved that a pathway of cell signaling initiating the apoptosis might be activated by these complexes, particularly in the case of complexes 1 and 2. The results of both UV-visible and CD measurements showed the significant ability of these complexes to interact with DNA. While fluorescence data revealed that these complexes cannot enter DNA structure by intercalation, molecular docking assessment proved their DNA groove binding ability. CONCLUSION: The remarkable apoptosis inducing activity of the binuclear platinum complexes 1 and 2 and their considerable interaction with DNA suggest them as the potential antitumor medicines.

Molecular interaction of fibrinogen with zeolite nanoparticles.

Fibrinogen is one of the key proteins that participate in the protein corona composition of many types of nanoparticles (NPs), and its conformational changes are crucial for activation of immune systems. Recently, we demonstrated that the fibrinogen highly contributed in the protein corona composition at the surface of zeolite nanoparticles. Therefore, understanding the interaction of fibrinogen with zeolite nanoparticles in more details could shed light of their safe applications in medicine. Thus, we probed the molecular interactions between fibrinogen and zeolite nanoparticles using both experimental and simulation approaches. The results indicated that fibrinogen has a strong and thermodynamically favorable interaction with zeolite nanoparticles in a non-cooperative manner. Additionally, fibrinogen experienced a substantial conformational change in the presence of zeolite nanoparticles through a concentration-dependent manner. Simulation results showed that both E- and D-domain of fibrinogen are bound to the EMT zeolite NPs via strong electrostatic interactions, and undergo structural changes leading to exposing normally buried sequences. D-domain has more contribution in this interaction and the C-terminus of γ chain (γ377-394), located in D-domain, showed the highest level of exposure compared to other sequences/residues.

Theranostic α-Lactalbumin-Polymer-Based Nanocomposite as a Drug Delivery Carrier for Cancer Therapy.

A nanotheranostic system was developed using α-lactalbumin along with Fe3O4 nanoparticles as an magnetic resonance imaging (MRI) contrast agent for medical imaging and doxorubicin as the therapeutic agent. α-lactalbumin was precipitated and cross-linked using poly(ethylene glycol) and glutaraldehyde. Besides, polyethylenimine was applied to increase the number of amine groups during cross-linking between α-lactalbumin and Fe3O4 nanoparticles. Interestingly, 90% of the initial protein used for the coaggregation process was incorporated in the prepared 130 nm nanocomposites, which facilitated the 85% doxorubicin loading. Formation of pH-sensitive imine bonds between glutaraldehyde and amine groups on α-lactalbumin and polyethylenimine resulted in higher release of doxorubicin at acidic pHs and consequently development of a pH-sensitive nanocarrier. The designed nanocomposite was less immunogenic owing to stimulating the production of less amounts of C3a, C5a, platelet factor 4, glycoprotein IIb/IIIa, platelet-derived ß-thromboglobulin, interleukin-6, and interleukin-1ß compared to the free doxorubicin. Furthermore, 1000 µg/mL nanocomposite led to 0.2% hemolytic activity, much less than the 5% standard limit. The void nanocarrier induced no significant level of cytotoxicity in breast cancer and normal cells following 96 h incubation. The doxorubicin-loaded nanocomposite presented higher cytotoxicity, apoptosis induction, and doxorubicin uptake in cancer cells than free doxorubicin. Conversely, lower cytotoxicity, apoptosis induction, and doxorubicin uptake were observed in normal cells treated with the doxorubicin-loaded nanocarrier compared to free doxorubicin. In line with the results of in vitro experiments, in vivo studies on tumor-bearing mice showed more suppression of tumor growth by the doxorubicin-loaded nanocomposite compared to the free drug. Moreover, the pharmacokinetic study revealed slow release of doxorubicin from the nanocomposite. Besides, in vitro and in vivo MRI studies presented a higher r2/r1 ratio and comparable contrast to the commercially available DOTAREM, respectively. Our findings suggest that this new nanocomposite is a promising nanotheranostic system with promising potential for cancer therapy and diagnosis.

A biophysical study on the mechanism of interactions of DOX or PTX with α-lactalbumin as a delivery carrier.

Doxorubicin and paclitaxel, two hydrophobic chemotherapeutic agents, are used in cancer therapies. Presence of hydrophobic patches and a flexible fold could probably make α-Lactalbumin a suitable carrier for hydrophobic drugs. In the present study, a variety of thermodynamic, spectroscopic, computational, and cellular techniques were applied to assess α-lactalbumin potential as a carrier for doxorubicin and paclitaxel. According to isothermal titration calorimetry data, the interaction between α-lactalbumin and doxorubicin or paclitaxel is spontaneous and the K (M-1) value for the interaction of α-lactalbumin and paclitaxel is higher than that for doxorubicin. Differential scanning calorimetry and anisotropy results indicated formation of α-lactalbumin complexes with doxorubicin or paclitaxel. Furthermore, molecular docking and dynamic studies revealed that TRPs are not involved in α-Lac's interaction with Doxorubicin while TRP 60 interacts with paclitaxel. Based on Pace analysis to determine protein thermal stability, doxorubicin and paclitaxel induced higher and lower thermal stability in α-lactalbumin, respectively. Besides, fluorescence lifetime measurements reflected that the interaction between α-lactalbumin with doxorubicin or paclitaxel was of static nature. Therefore, the authors hypothesized that α-lactalbumin could serve as a carrier for doxorubicin and paclitaxel by reducing cytotoxicity and apoptosis which was demonstrated during our in vitro cell studies.

Protective effects of silibinin on insulin amyloid fibrillation, cytotoxicity and mitochondrial membrane damage.

A growing body of evidence suggests that secretion and assembly of insulin to amyloid fibrils reduce its efficacy in treating type II diabetes and may lead to dysfunctioning of several organs. The research presented here explores the effects of silibinin on the in vitro amyloid fibrillation and cytotoxicity of bovine insulin fibrils on SH-SY5Y human neuroblastoma cells. Interaction of the resulting structures with rat brain mitochondria was also investigated. Using a range of methods for amyloid detection we showed that insulin fibrillation was significantly inhibited by silibinin in a dose-dependent fashion. Moreover, we found that silibinin was very effective in attenuating insulin fibril-induced neuronal toxicity characterized by decrease of cell viability, the release of lactate dehydrogenase, intracellular reactive oxygen species enhancement, morphological alterations, and apoptotic cell death induction. While insulin fibrillation products showed the capacity to damage mitochondria, the resultant structures produced in the presence of silibinin were totally ineffective. Together, results demonstrate the capacity of insulin fibrils to cause SH-SY5Y cell death by inducing necrosis/apoptosis changes and suggest how silibinin may afford protection. It is concluded that elucidation of such protection may provide important insights into the development of preventive and therapeutic agents for amyloid-related diseases.

Paclitaxel inhibited lysozyme fibrillation by increasing colloidal stability through formation of "off-pathway" oligomers.

Protein fibrillation is a challenging issue in medicine, causing many diseases, and an impediment to pharmaceutics and protein industry. Many chemicals, especially polyphenol compounds and aromatic small molecules, have been widely used as an effective strategy to combat protein fibril formation. Hence, understanding mechanisms of fibrillation inhibition and contributing forces in this process are significant. In this study, the inhibitory effect of paclitaxel on lysozyme fibrillation was investigated with respect to thermal and colloidal stability. Fibrillation was monitored with ThT fluorescence, circular dichroism, and AFM; paclitaxel-lysozyme interaction with isothermal titration calorimetry and docking; thermal and colloidal stability with differential scanning calorimetry and zeta-pulse, respectively. Paclitaxel inhibited lysozyme fibrillation, and interacted with lysozyme through hydrogen bonds and van der Waals' interactions. The viability of PC12 cells retrieved as a result of fibrillation inhibition by paclitaxel. Hydrophobic forces dominantly shielded the aggregation-prone region of lysozyme and suppressed the effective interactions between lysozyme monomers. Although paclitaxel did not affect lysozyme's thermal stability, it increased lysozyme's colloidal stability by either increasing the surface charge density or charge distribution on lysozyme. In conclusion, our results suggest a model for paclitaxel's inhibitory role through two complementary steps driving to "off-pathway" oligomer formation and attenuation of fibril formation.

Unraveling the novel effects of aroma from small molecules in preventing hen egg white lysozyme amyloid fibril formation.

This study investigated for the first time the molecular effectiveness of 'aroma' from three small molecules including a phenol (phenyl ethyl alcohol; PEA) and an aldehyde (cinnamaldehyde; Cin) both containing an aromatic ring, and a diamine (N,N,N,N'- Tetramethylethylenediamine; TEMED) at two different amounts (small; S and large; L) in preventing hen egg white lysozyme (HEWL) amyloid fibril formation using Thioflavin T and Nile red fluorescence assays, circular dichroism spectroscopy, SDS-polyacrylamide gel electrophoresis, atomic force microscopy, dynamic light scattering and HEWL activity test. Interestingly, the results revealed that (1) the aroma of PEA, identified as an active constituent of Rosa damascena, prevented fibril formation since PEA-L was able to trap the oligomeric form of HEWL in contrast to PEA-S where protofibrils but not mature fibrils were formed; (2) Cin, previously shown to prevent fibril formation in the liquid form, was also shown to do so in the aroma form by producing protofibrils and not mature fibrils in both Cin- L and Cin-S aroma forms and (3) the aroma of TEMED-L was able to retain HEWL's native structure completely and prevented both aggregation and fibril formation, while TEMED-S prevented HEWL fibril formation and instead directed the pathway towards amorphous aggregate formation. Furthermore, the ability to trap oligomeric species (by PEA-L aroma) is of great importance for further research as it provides routes for preventing the formation of toxic oligomeric intermediates along the fibrillation pathway. Last but not least, the novelty of this in vitro study on the effect of aroma at the molecular level with a unique experimental set-up using HEWL as a model protein in assessing amyloid fibril formation paves the way for more and detailed studies on the importance of aroma producing molecules and their effects.