Impact of Oxysterols in Age-Related Disorders and Strategies to Alleviate Adverse Effects.

Oxysterols or cholesterol oxidation products are a class of molecules with the sterol moiety, derived from oxidative reaction of cholesterol through enzymatic and non-enzymatic processes. They are widely reported in animal-origin foods and prove significant involvement in the regulation of cholesterol homeostasis, lipid transport, cellular signaling, and other physiological processes. Reports of oxysterol-mediated cytotoxicity are in abundance and thus consequently implicated in several age-related and lifestyle disorders such as cardiovascular diseases, bone disorders, pancreatic disorders, age-related macular degeneration, cataract, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and some types of cancers. In this chapter, we attempt to review a selection of physiologically relevant oxysterols, with a focus on their formation, properties, and roles in health and disease, while also delving into the potential of natural and synthetic molecules along with bacterial enzymes for mitigating oxysterol-mediated cell damage.

Thermal ablation in pancreatic cancer: A scoping review of clinical studies.

Background: Pancreatic cancer is a deadly cancer with a 5-year survival rate less than 10%. Only 20% of patients are eligible to receive surgery at diagnosis. Hence, new therapies are needed to improve outcomes for non-surgical candidates. Thermal ablation techniques can offer a non-invasive alternative to surgery. Aim: The aim of this review is to map the literature for the use of thermal ablative techniques: Radiofrequency ablation (RFA), High-intensity focused ultrasound (HIFU), Microwave ablation (MWA), and Laser ablation (LA) in the management of patients with PC. Methods: A search strategy was applied to PUBMED and EMBASE using keywords concerning pancreatic cancer, radiofrequency ablation, ultrasound ablation, laser ablation, and microwave ablation. The studies that fit this inclusion criteria were summarized in table format and results reviewed for interpretation. Results: 72 clinical studies were included. Most of the included studies related to RFA (n=35) and HIFU (n=27). The most common study design was retrospective (n=33). Only 3 randomized control trials (RCT) were included, all of which related to RFA. Safety outcomes were reported in 53 of the 72 studies, and survival outcomes were reported in 39. Statistically significant survival benefits were demonstrated in 11 studies. Conclusion: The evidence for the benefit of MWA and LA in PC patients is limited. RFA and HIFU are safe and feasible therapies to be used in PC patients. Further RCTs where thermal techniques are standardized and reported are necessary in the future to elucidate thermal ablation's clinical utility, and before an evidence-based decision on its routine use in PC management can be considered.

Use of gold nanoparticle-silibinin conjugates: A novel approach against lung cancer cells.

Lung cancer presents one of the most challenging carcinomas with meager 5-year survival rates (less than 20%), high metastasis and high recurrence due to chemo- and radio- resistance. An alternative or complementation to existing prognosis modalities is the use of phytochemicals such as silibinin, which targets essential cytokines, angiogenic factors and transcription factors for a profound anti-tumor effect. However, the problems of low solubility in an aqueous physiological environment, poor penetration, high metabolism and rapid systemic clearance limit the therapeutic use of silibinin. Conjugation of gold nanoparticles (GNPs) with silibinin may overcome the above challenges along with distinct advantages of biocompatibility, optical properties for monitoring and causation of cytotoxicity in cancer cells. The current study thus aims to develop silibinin conjugated gold nanoparticles (Sb-GNPs) with pH responsive release in the cancer microenvironment, optimizing several parameters for its higher activity and further evaluate the nanoplatform for their efficacy in inducing cell death in vitro against A549 lung cancer cells. GNPs was synthesized using trisodium citrate dihydrate as the reducing agent and further used for the conjugation of silibinin. The synthesized GNPs were found to be monodispersed and spherical in shape. The silibinin was successfully conjugated with gold nanoparticles and long-term stability of GNPs and Sb-GNPs nanoconjugates in suspension phase was confirmed by FTIR and DLS. Anticancer properties of Sb-GNPs were confirmed by different assay using MTT, Trypan blue dye exclusion assay and cell cycle analysis assay. After conjugation of silibinin with GNPs, the efficacy of silibinin increased 4-5 times in killing the cancer cells. This is the first report on using silibinin gold nanoconjugate system for lung cancer therapy with promising future applications.

Microbial Nano-Factories: Synthesis and Biomedical Applications.

In the recent times, nanomaterials have emerged in the field of biology, medicine, electronics, and agriculture due to their immense applications. Owing to their nanoscale sizes, they present large surface/volume ratio, characteristic structures, and similar dimensions to biomolecules resulting in unique properties for biomedical applications. The chemical and physical methods to synthesize nanoparticles have their own limitations which can be overcome using biological methods for the synthesis. Moreover, through the biogenic synthesis route, the usage of microorganisms has offered a reliable, sustainable, safe, and environmental friendly technique for nanosynthesis. Bacterial, algal, fungal, and yeast cells are known to transport metals from their environment and convert them to elemental nanoparticle forms which are either accumulated or secreted. Additionally, robust nanocarriers have also been developed using viruses. In order to prevent aggregation and promote stabilization of the nanoparticles, capping agents are often secreted during biosynthesis. Microbial nanoparticles find biomedical applications in rapid diagnostics, imaging, biopharmaceuticals, drug delivery systems, antimicrobials, biomaterials for tissue regeneration as well as biosensors. The major challenges in therapeutic applications of microbial nanoparticles include biocompatibility, bioavailability, stability, degradation in the gastro-intestinal tract, and immune response. Thus, the current review article is focused on the microbe-mediated synthesis of various nanoparticles, the different microbial strains explored for such synthesis along with their current and future biomedical applications.

Mechanistic Aspects of Microbe-Mediated Nanoparticle Synthesis.

In recent times, nanoparticles (NPs) have found increasing interest owing to their size, large surface areas, distinctive structures, and unique properties, making them suitable for various industrial and biomedical applications. Biogenic synthesis of NPs using microbes is a recent trend and a greener approach than physical and chemical methods of synthesis, which demand higher costs, greater energy consumption, and complex reaction conditions and ensue hazardous environmental impact. Several microorganisms are known to trap metals in situ and convert them into elemental NPs forms. They are found to accumulate inside and outside of the cell as well as in the periplasmic space. Despite the toxicity of NPs, the driving factor for the production of NPs inside microorganisms remains unelucidated. Several reports suggest that nanotization is a way of stress response and biodefense mechanism for the microbe, which involves metal excretion/accumulation across membranes, enzymatic action, efflux pump systems, binding at peptides, and precipitation. Moreover, genes also play an important role for microbial nanoparticle biosynthesis. The resistance of microbial cells to metal ions during inward and outward transportation leads to precipitation. Accordingly, it becomes pertinent to understand the interaction of the metal ions with proteins, DNA, organelles, membranes, and their subsequent cellular uptake. The elucidation of the mechanism also allows us to control the shape, size, and monodispersity of the NPs to develop large-scale production according to the required application. This article reviews different means in microbial synthesis of NPs focusing on understanding the cellular, biochemical, and molecular mechanisms of nanotization of metals.

Phytochemical delivery through nanocarriers: a review.

In recent times, phytochemicals encapsulated or conjugated with nanocarriers for delivery to the specific sites have gained considerable research interest. Phytochemicals are mostly plant secondary metabolites which reported to be beneficial for human health and in disease theraphy. However, these compound are large size and polar nature of these compounds, make it difficult to cross the blood-brain barrier (BBB), endothelial lining of blood vessels, gastrointestinal tract and mucosa. Moreover, they are enzymatically degraded in the gastrointestinal tract. Therefore, encapsulation or conjugation of these compounds with nanocrriers could be an alternate way to enhance their bioefficacy by influencing their gastrointestinal stability, rate of absorption and dispersion. This review presents an overview of nanocarriers alternatives which improves therapeutic value and avoid toxicity, by releasing bioactive compounds specifically at target tissues with enhanced stability and bioavailability. Future investigations may emphasize on deciphering the structural changes in nanocarriers during digestion and absorption, the difference between in-vitro and in-vivo digestion simulations, and impact of nanocarriers on the metabolism of phytochemicals.

Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase.

Fanconi anemia (FA) is a unique DNA damage repair pathway. To date, 22 genes have been identified that are associated with the FA pathway. A defect in any of those genes causes genomic instability, and the patients bearing the mutation become susceptible to cancer. In our earlier work, we identified that Fanconi anemia protein G (FANCG) protects the mitochondria from oxidative stress. In this report, we have identified eight patients having a mutation (C.65G>C), which converts arginine at position 22 to proline (p.Arg22Pro) in the N terminus of FANCG. The mutant protein, hFANCGR22P, is able to repair the DNA and able to retain the monoubiquitination of FANCD2 in the FANCGR22P/FGR22P cell. However, it lost mitochondrial localization and failed to protect mitochondria from oxidative stress. Mitochondrial instability in the FANCGR22P cell causes the transcriptional downregulation of mitochondrial iron-sulfur cluster biogenesis protein frataxin (FXN) and the resulting iron deficiency of FA protein FANCJ, an iron-sulfur-containing helicase involved in DNA repair.

7-Ketocholesterol and 7ß-hydroxycholesterol: In vitro and animal models used to characterize their activities and to identify molecules preventing their toxicity.

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7ß-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7ß-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

Stability of Therapeutic Enzymes: Challenges and Recent Advances.

Enzymes are biocatalysts that have found profound applications in the current biotherapeutic industry and play a crucial role in diagnosis, prevention, and biochemical analysis of major diseases. However, stability, protein degradation and immunogenicity in the body present unique challenges that are faced upon sustained use of such enzymes. The present chapter is an attempt to dissect the state-of-the-art in relation to the challenges of development of therapeutic enzymes and the recent advances to address them. At the very outset, diseases where enzymes have found effective applications and the various causes of enzyme instability have been discussed. In recent times, polymer or nano- conjugated resistant delivery methods, as well as mutagenesis have led to manifold increase in enzyme stability against thermal denaturation, acidic gut environment, proteolysis and immunogenicity. Further, methods of analytical characterization of proteins have been highlighted and explored to shape future research directions.

Refolding of thermally denatured cholesterol oxidases by magnetic nanoparticles.

Proteins are prone to unfolding and subsequent denaturation by changes in temperature, pH and other harsh conditions. Nanoparticles act as artificial 'chaperones' due to favourable orientation of the proteins on their scaffold which prevents aggregation and reconfigures denatured proteins into their native functional state. In the present study, thermal denaturation of Cholesterol oxidases from Pseudomonas aeruginosa PseA, Rhodococcus erythropolis MTCC 3951 and Streptomyces sp. were studied at temperatures 50-70 °C. Further, these thermally denatured proteins were refolded using functionalized Magnetic Iron (II, III) oxide nanoparticles which was confirmed using DLS, Zeta Potential Measurements, fluorescence and CD spectroscopy. The refolded proteins were found to regain their secondary structure and activity to a great extent.

Cholesterol-oxidase-magnetic nanobioconjugates for the production of 4-cholesten-3-one and 4-cholesten-3, 7-dione.

Cholesterol oxidase(ChOx) enzyme isolated from Pseudomonas aeruginosa PseA(ChOxP) and Rhodococcus erythropolis MTCC 3951(ChOxR) strains as well as a commercial variant produced by Streptomyces sp.(ChOxS) were immobilized on silane modified iron(II, III)oxide magnetic nanoparticles(MNP) by covalent coupling methods. The nanobiocatalysts in case of ChOxP, ChOxR and ChOxS, retained 71, 91 and 86% of cholesterol oxidase activity respectively, as compared to their soluble counterparts. The catalytic efficiency of the immobilized enzymes on nanoparticles was more than 2.0 times higher than the free enzyme. They also showed enhanced pH and thermal stability. After 10 cycles of operation, the MNP-bioconjugates retained 50, 52 and 51% of residual activity in case of ChOxP, ChOxR and ChOxS respectively. The presence of enzyme on nanoparticles was confirmed by FTIR, SEM and TEM. The nanobiocatalysts were used for the biotransformation of cholesterol and 7-ketocholesterol to 4-cholesten-3-one and 4-cholesten-3, 7-dione respectively, which are industrially and medically important steroid precursors.

Biodegradation of 7-Ketocholesterol by Rhodococcus erythropolis MTCC 3951: Process optimization and enzymatic insights.

The oxidation of cholesterol results in the formation of oxysterols such as 7-ketocholesterol (7KC), which are implicated in a number of age-related disorders such as atherosclerosis, Alzheimers' disease and macular degeneration. Current modalities use antioxidants and other natural or synthetic molecules to reduce 7KC-induced cytotoxity. The alternative application of enzymes from microbial sources to degrade oxysterols in vitro and in vivo is an innovative approach. The present study aims to assess the potential of the bacteria Rhodococcus erythropolis MTCC 3951 in degrading 7KC and mining relevant enzymes involved. This strain has been previously reported to be a degrader of xenobiotics such as polyphenols, toluene and catechol. Under optimized conditions, Rhodococcus erythropolis MTCC 3951 is found to degrade 93% of 1g/l concentration of 7KC within 15days of incubation. The extra- and intra-cellular extracts were also able to hydrolyse the compound indicating the involvement of enzymatic systems in the process. The strain produced cholesterol oxidase, lipase, dehydrogenase and reductase in the presence of 7KC. We have also identified a few intermediate products to predict the degradation pathway.

Biodegradation of cytotoxic 7-Ketocholesterol by Pseudomonas aeruginosa PseA.

The present study aims to degrade 7-Ketocholesterol (7KC), a major oxysterol implicated in many age-related disorders, through microbial means and find candidate enzymes involved for further application in food systems and as a therapeutic. During initial screening of previously isolated bacteria from our laboratory, Pseudomonas aeruginosa PseA was found to be a potential degrader strain using 7KC as a sole carbon source. Under optimized conditions, it is able to degrade 88% of an initial concentration of 1000ppm (1g/l) 7KC. Preliminary in vitro studies with extra-cellular extract has shown degradation of the compound, thus reinforcing the occurrence of suitable enzymatic systems involved in the process. We have been able to identify cholesterol oxidase as one such potential enzyme. Some intermediate products of degradation have also been identified. This is the first detailed report of 7KC degradation by a P. aeruginosa strain.