Nanosilver reinforced Parmelia sulcata extract efficiently induces apoptosis and inhibits proliferative signalling in MCF-7 cells.

The Lichen, Parmelia sulcata synthesizes various secondary metabolites, in which phenolic based compounds received much attention due to their importance in biomedical application. Especially the phenolic compound was effective against the cancer treatment. An effective administration of such plant natural product can represent a significant conventional management of cancer in terms of chemoprevention. The nanomedicines are group of agents that selectively interfere the cancer cells which leads to reduction of side effect thereby reducing the doses. Silver nanoparticles is a promising antitumor agent, however, the conventional production of silver nanoparticles have many drawbacks which led to increase in need of eco-friendly biological production methods. In this study, we made an attempt to synthesise a nano silver (Ps-AgNPs) from phenolic extract of lichen Parmelia sulcata extract. The Ps-AgNps was applied for anticancer activity using MCF-7 cells and the effect was characterised by western blotting method. The FTIR, XRD, UV and TEM results confirms the presence of silver nanoparticles in phenolic extract of lichen Parmelia sulcata. The cytotoxicity assay shows that the Ps-AgNPs is toxic against cancer cells (MCF-7) but not to normal cells (NIH3T3), which confirm the selective induction of cell death (apoptosis) against cancer cells. The Western blot analysis also clearly indicates the down regulation of inflammatory genes (TNF-alpha and IL-6) and cell cycle genes (PCNA and Cyclin-D1) thus promoting intrinsic apoptotic pathway. The results suggest that Ps-AgNPs can effectively kill cancer cells and can be used as an alternative therapeutic agent for cancer treatment.

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum).

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20 mM tris-CaCl2 buffer (pH 8.2) with a flow rate of 0.5 mL min-1. The molecular weight and purity of ∼23 kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697 U mg-1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.

Inter-domain helix h10DOMI-h1DOMII is important in the molecular interaction of bovine serum albumin with curcumin: spectroscopic and computational analysis.

The importance of domain II in the molecular interaction of bovine serum albumin (BSA) with curcumin was investigated by fluorescence spectroscopy and molecular docking. At pH 7.4 BSA is in its native state. Domain III of BSA unfolds at pH 4.0, and domains I and III unfold in the presence of 5 M urea. Curcumin has a high quenching constant (K SV ~ 10(4) M (-1)) and moderate binding affinity (n ~ 0.5). The standard free energy change (∆G° ~ -25 kJ mol(-1)) indicates that binding is spontaneous. No significant change in ∆G° observed after unfolding of domain I or domain III. The standard change in enthalpy (∆H°) and entropy (∆S°) show that ionic and hydrophobic interactions are important in the binding. Computational studies revealed that the inter-domain helix h10DOMI-h1DOMII of BSA is the region of binding of curcumin, and residues Arg198 and Arg208 are important in binding. The binding site is located between sub-domains IB and IIA, and overlaps drug binding site-1.

Recent Developments of Hybrid Fluorescence Techniques: Advances in Amyloid Detection Methods.

Amyloid fibrils are formed from various pathological proteins. Monitoring their aggregation process is necessary for early detection and treatment. Among the available detection techniques, fluorescence is simple, intuitive, and convenient due to its sensitive and selective mode of detection. It has certain disadvantages like poor photothermal stability and detection state limitation. Research has focused on minimising the limitation by developing hybrid fluorescence techniques. This review focuses on the two ways fluorescence (intrinsic and extrinsic) has been used to monitor amyloid fibrils. In intrinsic/label free fluorescence: i) The fluorescence emission through aromatic amino acid residues like phenylalanine (F), tyrosine (Y) and tryptophan (W) is present in amyloidogenic peptides/protein sequence. And ii) The structural changes from alpha helix to cross-ß-sheet structures during amyloid formation contribute to the fluorescence emission. The second method focuses on the use of extrinsic fluorophores to monitor amyloid fibrils i) organic dyes/small molecules, ii) fluorescent tagged proteins, iii) nanoparticles, iv) metal complexes and v) conjugated polymers. All these fluorophores havetheir own limitations. Developing them into hybrid fluorescence techniques and converting it into biosensors can contribute to early detection of disease.

Recent Advancements in Biosensors for the Detection and Characterization of Amyloids: A Review.

Modern medicine has increased the human lifespan. However, with an increase in average lifespan risk of amyloidosis increases. Amyloidosis is a condition characterized by protein misfolding and aggregation. Early detection of amyloidosis is crucial, yet conventional diagnostic methods are costly and lack precision, necessitating innovative tools. This review explores recent advancements in diverse amyloid detection methodologies, highlighting the need for interdisciplinary research to develop a miniaturized electrochemical biosensor leveraging nanotechnology. However, the diagnostics industry faces obstacles such as skilled labor shortages, standardized selection processes, and concurrent multi-analyte identification challenges. Research efforts are focused on integrating electrochemical techniques into clinical applications and diagnostics, with the successful transition of miniaturized technologies from development to testing posing a significant hurdle. Label-free transduction techniques like voltammetry and electrochemical impedance spectroscopy (EIS) have gained traction due to their rapid, cost-effective, and user-friendly nature.

At very low concentrations known chaotropes act as kosmotropes for the N and B isoforms of human serum albumin.

Very few studies have been done to understand the effect of millimolar concentrations of chaotropes on protein structure. In our previous study we observed that the secondary and tertiary structure of human serum albumin (HSA) increases in the presence of 5 mmol/L urea. Micelle formation in amphoteric detergents increases in the presence of equivalent concentrations of urea. Here, we observed a significant increase in the secondary and tertiary structure of HSA. Interestingly, guanidine hydrochloride, another chaotropic agent, also shows a similar effect. Our results show electrostatic interaction may play a role in neutral to basic transition in HSA. This study further supports the claim that at millimolar concentrations the chaotropes may act as kosmotropes for proteins.

Picloram binds to the h1 and h4 helices of HSA domain IIIA at drug binding site 2.

Picloram (PC) is a systemic herbicide that controls herbaceous weeds and woody plants. HSA, the most abundant protein in human physiology, binds to all exogenic and endogenic ligands. PC is a stable molecule (t1/2∼157-513 days) and a potential threat to human health via the food chain. HSA and PC binding study has been done to decipher the location and thermodynamics of binding. It has been studied with prediction tools like autodocking and MD simulation and then confirmed with fluorescence spectroscopy. HSA fluorescence was quenched by PC at pH 7.4 (N state), pH 3.5 (F state), and pH 7.4 with 4.5 M urea (I state) at temperatures 283 K, 297 K, and 303 K. The location of binding was found to be interdomain between II and III which overlaps with drug binding site 2. The binding was spontaneous, and entropy-driven that show a noticeable increase in binding with the increase in temperature. No secondary structure change at the native state has been observed due to binding. The binding results are important to understand the physiological assimilation of PC. In silico predictions and the results of spectroscopic studies unambiguously indicate the locus and nature of the binding.

Interaction Mechanism between α-Lactalbumin and Caffeic Acid: A Multispectroscopic and Molecular Docking Study.

Caffeic acid (CA) is a phenolic acid found in a variety of foods. In this study, the interaction mechanism between α-lactalbumin (ALA) and CA was explored with the use of spectroscopic and computational techniques. The Stern-Volmer quenching constant data suggest a static mode of quenching between CA and ALA, depicting a gradual decrease in quenching constants with temperature rise. The binding constant, Gibbs free energy, enthalpy, and entropy values at 288, 298, and 310 K were calculated, and the obtained values suggest that the reaction is spontaneous and exothermic. Both in vitro and in silico studies show that hydrogen bonding is the dominant force in the CA-ALA interaction. Ser112 and Lys108 of ALA are predicted to form three hydrogen bonds with CA. The UV-visible spectroscopy measurements demonstrated that the absorbance peak A280nm increased after addition of CA due to conformational change. The secondary structure of ALA was also slightly modified due to CA interaction. The circular dichroism (CD) studies showed that ALA gains more α-helical structure in response to increasing concentration of CA. The surface hydrophobicity of ALA is not changed in the presence of ethanol and CA. The present findings shown herein are helpful in understanding the binding mechanism of CA with whey proteins for the dairy processing industry and food nutrition security.

Hexametaphosphate, a Common Food Additive, Aggregated the Hen Egg White Lysozyme.

Polyphosphate polymers are chains of phosphate monomers chemically bonded together via phosphoanhydride bonds. They are found in all prokaryotic and eukaryotic organisms and are among the earliest, most anionic, and most mysterious molecules known. They are everywhere, from small cellular components to additives in our food. There is a strong association between hyperphosphatemia and mortality. That is why it is crucial to assess how polyphosphates, as food additives, affect the quality of edible proteins. This study investigated the effect of inexpensive and widely used food additives (hexametaphosphate labeled as E452) on bakery items, meat products, fish, and soft drinks. Using various spectroscopic and microscopic techniques, we examined how hexametaphosphate affected the aggregation propensity, structure, and stability of a commonly used food protein: hen egg white lysozyme (HEWL). The solubility of HEWL is affected in a bimodal fashion by the concentration of hexametaphosphate. The bimodal concentration-dependent effect was also observed in the tertiary and secondary structural changes. Hexametaphosphate-induced HEWL aggregates were amorphous, as evidenced by ThT fluorescence, far-UV CD, and TEM imaging. This study showed that the food additive (hexametaphosphate) may denature and aggregate proteins and may lead to undesirable health issues.

Expression of a Tagless Single-Chain Variable Fragment (scFv) of Anti-TNF-α by a Salt Inducible System and its Purification and Characterization.

BACKGROUND: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases, such as rheumatoid arthritis and Crohn's disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb's auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α. OBJECTIVE: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer. METHODS: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out. RESULTS: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ~ 28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry. CONCLUSION: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.

Ligand binding strategies of human serum albumin: how can the cargo be utilized?

Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications.

Deciphering the role of premicellar and micellar concentrations of sodium dodecyl benzenesulfonate surfactant in insulin fibrillation at pH 2.0.

Amyloid fibril formation by proteins and their deposition in cells and tissues are associated with several amyloid-based disorders. Understanding the mechanism of amyloid fibril formation is thus of the utmost importance for the designing ligands that could prevent or inhibit the fibrillation process and help to treat of such disorders. We describe the stimulatory effect of sodium dodecyl benzenesulfonate (SDBS) on insulin amyloid fibrillation at pH 2.0 and the characterization of SDBS-induced insulin aggregation using spectroscopy and microscopy. We found that SDBS induced amyloid-like aggregates of insulin at sub-micellar (0.1-1.2 mM), but not post-micellar (≥2.0 mM) concentrations. The amyloid fibrillation of insulin induced by SDBS was kinetically rapid and escaped the lag phase. Far-UV CD findings suggested that the α-helical content of insulin transformed into cross-ß structure and mixed α and ß structures when incubated with sub-micellar and post-micellar SDBS concentrations, respectively. The overall results indicated that low, but not high SDBS concentrations induce amyloid-like insulin aggregates and fibrils.

Streptozocin; a GLUT2 binding drug, interacts with human serum albumin at loci h6DOM3-h7DOM3.

Streptozocin (STZ) is a broad range antibiotic, highly genotoxic, antineoplastic and hyperglycemic. HSA is the most abundant protein in physiology and it binds to almost all exogenic and endogenic ligands, including drugs. STZ-induced fluorescence quenching of HSA has been done at pH 7.4, pH 3.5 and at pH 7.4 with 4.5 M urea at temperatures 286 K, 291 K, and 306 K. Ksv found to be 103 M-1, binding constant 1.5X103M-1 and binding sites ~1. But, Ksv for HSA and glucopyranose interaction was found lesser than that of HSA-STZ binding. Binding of STZ/glucopyranose on HSA seems to result in complex formation as calculated Kq > 1010 M-1 s-1. The number of binding sites, binding constants, and binding energies were increased with temperature. The ΔG0, ΔH0, and ΔS0 for HSA-STZ interaction were found to be -17.7 × 103 J·mol-1; 2.34 × 105 J·mol-1 and 841 JK-1 mol-1 respectively at pH 7.4 and 291 K. The comparative bindings of N, F and I states of HSA with STZ and their molecular docking analyses indicate that IIIA-B junction (i.e., inter-helix h6DOM3-h7DOM3) is the probable binding site, a locus close to fatty acid binding site-5. These results could be useful for therapeutic and analytical exploitation of STZ, as albumin used as the vehicle for drug delivery.

Different conformational states of hen egg white lysozyme formed by exposure to the surfactant of sodium dodecyl benzenesulfonate.

The aim of this study was to investigate the effects of sodium dodecyl benzenesulfonate (SDBS) on hen egg white lysozyme (HEWL) fibrillogenesis at pH 7.4. HEWL fibrillogenesis in the presence of SDBS was characterized using several spectroscopic techniques (turbidity, light scattering, intrinsic fluorescence, ThT binding assay, ThT kinetics, far-UV CD, and transmission electron mmicroscopy). The turbidity and light scattering data revealed that SDBS induces aggregation in HEWL in dose-dependent manner. HEWL aggregation was seen at low SDBS concentrations (0.03 to 0.5 mM) but it was not observed at concentrations of SDBS at >0.6 mM. The ThT and TEM data clearly showed that the aggregates formed in the presence of SDBS had an amyloid-like morphology. From the CD analysis it was clear that low SDBS concentrations decreases the α-helical content while the ß-sheet content increased. As the SDBS concentration further increased, the α-helical content increased again. The ThT kinetics analysis revealed that the HEWL monomer directly converted into the amyloid fibril without lag phase. All the spectroscopic and microscopic results support the finding that low concentrations of SDBS stimulate fibrillogenesis in HEWL, and that no fibrillogenesis occurs at higher SDBS concentrations.

Allura red rapidly induces amyloid-like fibril formation in hen egg white lysozyme at physiological pH.

Allura red (AR) is an artificial azo dye mostly used in food industries and has potential health risks. We examined the role of AR in amyloidogenesis using hen egg white lysozyme (HEWL) at pH 7.0. The amyloidogenic induction properties of AR in HEWL were identified by circular dichroism (CD), turbidity, intrinsic fluorescence, light scattering, transmission electron microscopy (TEM), and molecular dynamic simulation studies. Turbidity and light scattering measurements showed that HEWL becomes aggregated in the presence of 0.03-15.0 mM of AR at pH 7.0 but not at very low AR concentrations (0.01-0.28 mM). However, AR-induced aggregation is a kinetically rapid process, with no observable lag phase and saturation within 6 s. The kinetics results suggested that the HEWL aggregation induced by AR is very rapid. The CD results demonstrated that the total ß-sheet content of HEWL was increased in the AR treated samples. The TEM results are established that AR-induced aggregates had amyloid-like structures. Molecular dynamics simulations analysis showed that the bound AR-HEWL structures were highly favored compared to unbound structures. The mechanism of AR-induced amyloid fibril formation may involve electrostatic, hydrogen bonding, and hydrophobic interactions.

A quercetin-based flavanoid (rutin) reverses amyloid fibrillation in ß-lactoglobulin at pH 2.0 and 358 K.

ß-lactoglobulin (BLG) is a well characterized milk protein and a model for folding and aggregation studies. Rutin is a quercetin based-flavanoid and a famous dietary supplement. It is a potential protector from coronary heart disease, cancers, and inflammatory bowel disease. In this study, amyloid fibrillation is reported in BLG at pH 2.0 and temperature 358 K. It is inhibited to some extent by rutin with a rate of 99.3 h-1 M-1. Amyloid fibrillation started taking place after 10 h of incubation and completed near 40 h at a rate of 16.6 × 10-3 h-1, with a plateau during 40-108 h. Disruption of tertiary structure of BLG and increased solvent accessibility of hydrophobic core seem to trigger intermolecular assembly. Increase in 7% ß-sheet structure at the cost of 10% α-helical structures and the electron micrograph of BLG fibrils at 108 h further support the formation of amyloid. Although it could not block amyloidosis completely, and even the time required to reach plateau remains the same, a decrease of growth rate from 16.6 × 10-3 to 13.5 × 10-3 h-1 was observed in the presence of 30.0 µM rutin. Rutin seems to block solvent accessibility of the hydrophobic core of BLG. A decrease in the fibril population was observed in electron micrographs, with the increase in rutin concentration. All evidences indicate reversal of fibrillation in BLG in the presence of rutin.

Characterization of fluoroalcohols-induced intermediates of Mucor miehei lipase at low pH.

We have previously characterized an acid-unfolded (U(A)) state of Mucor miehei lipase at pH 2. The effect of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) resulted in characterization of molten-globule (MG) like states with beta-sheet secondary structure at 15% (v/v) TFE and 6% (v/v) HFIP. alpha-Helical states accumulate at 80% (v/v) TFE and 30% (v/v) HFIP.

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study.

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0-20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.

Unraveling the molecular mechanism of the effects of sodium dodecyl sulfate, salts, and sugars on amyloid fibril formation in camel IgG.

Sodium dodecyl sulfate (SDS) is an anionic surfactant that can be used to stimulate protein fibrillation in vitro. Here, we investigated the effects of SDS on camel IgG aggregation at pH 3.5 and 7.4. SDS-induced amyloid fibril formation in camel IgG was examined by turbidity measurements, Rayleigh scattering, Thioflavin T (ThT) fluorescence, intrinsic fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM). The results suggest that low SDS concentrations (0.2-2.0 mM) induce amyloid-like aggregates of camel IgG at pH 3.5, indicating an SDS/camel IgG ratio below 1000. However, in the presence of higher concentrations of SDS (2.5-10.0 mM), amyloid fibril formation was not observed. Furthermore, at the higher concentrations, the ß-sheet structure of camel IgG was transformed into a α-helical structure. The amyloid fibril formation was not observed in the presence of SDS at pH 7.4. Additionally, the role of salts and sugars was evaluated in the SDS-induced aggregation process. Interestingly, in the presence of 0.15 N of NaCl and (NH4)2SO4, SDS promoted camel IgG aggregation up to very high concentrations of SDS (0.2-10.0 mM; SDS/camel IgG ratio, 95-4750) and no suppression was observed. Moreover, osmoprotectants (trehalose and sucrose) were ineffective, neither promoting nor inhibiting the SDS-induced aggregation process. However, at pH 3.5, electrostatic and hydrophobic interactions, and hydrogen bonds were the major contributing factors in SDS-induced fibrillation. However, no aggregation was observed at pH 7.4 due to electrostatic repulsion between SDS and camel IgG because both of these molecules have overall similar charges.

Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity.

Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical-scavenging activity in a concentration-dependent manner with IC50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC50) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.