Role of Exosomal MicroRNAs in Cell-to-Cell Communication.

Exosomes, a type of extracellular vesicle, are small vesicles (30-100 nm) secreted into extracellular space from almost all types of cells. Exosomes mediate cell-to-cell communication carrying various biologically active molecules including microRNAs. Studies have shown that exosomal microRNAs play fundamental roles in healthy and pathological conditions such as immunity, cancer, and inflammation. In this chapter, we introduce the current knowledge on exosome biogenesis, techniques used in exosome research, and exosomal miRNA and their functions in biological and pathological processes.

Quercetin in the form of a nano-antioxidant (QTiO2) provides stabilization of quercetin and maximizes its antioxidant capacity in the mouse fibroblast model.

Living cells are constantly exposed to reactive oxygen species (ROS) causing them to rely on a constant supply of exogenous antioxidants. Quercetin (Q) is one of the potent exogenous antioxidants utilized in various antioxidant formulations. However, the potential application of Q is largely limited because of its poor water solubility. In this study, we employed titanium dioxide (TiO2) nanoparticles to maximize cellular penetration and antioxidant effect of Q on mouse fibroblast cells. To accomplish this, polyethylene glycol (PEG) modified TiO2-nanoparticle surfaces were utilized that exhibited better dispersion, with enhanced biocompatibility. Cell viability assays using Q and Q-conjugated TiO2-nanoparticles (QTiO2) were evaluated in terms of cell morphology as well as with an immunoblotting analysis to look for key biomarkers of apoptosis. In addition, cleavages of Cas 3 and PARP were obtained in cells treated with Q. Furthermore, antioxidant defence with QTiO2 was validated by means of the Nrf2 upregulation pathway. We also observed increased expressions of target enzymes; HO-1, NQO1 and SOD1 in QTiO2-treated cells. The antioxidant potency of the QTiO2 nano-antioxidant form was successfully tested in ROS and superoxide radicals induced cells. Our results demonstrated that the QTiO2 nano-antioxidant promoted a high quercetin bioavailability and stability, in cells with maximal antioxidant potency against ROS, with no signs of cytotoxicity.

Isolation of large dense-core vesicles from bovine adrenal medulla for functional studies.

Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.

Role of mTORC1 and mTORC2 in Breast Cancer: Therapeutic Targeting of mTOR and Its Partners to Overcome Metastasis and Drug Resistance.

Based on the insights gleaned from decades of research, it seems clear that mechanistic target of rapamycin (mTOR) is an essential signaling node that integrates environmental clues for regulation of cell survival, metabolism and proliferation of the cells. However, overwhelmingly increasing scientific evidence has added a new layer of intricacy to already complicated and versatile signaling pathway of mTOR. Deregulation of spatio-temporally controlled mTOR-driven pathway played contributory role in breast cancer development and progression. Pharmacologists and molecular biologists have specifically emphasized on the identification and development of mTOR-pathway inhibitors. In this chapter we have attempted to provide an overview of the most recent findings related to therapeutic targeting of mTOR-associated mTORC1 and mTORC2 in breast cancer. We have also comprehensively summarized regulation of mTOR and its partners by microRNAs in breast cancer.

Augmented mitochondrial cytochrome c oxidase activity in children with iron deficiency: a tandem between iron and copper?

INTRODUCTION: Dyshomeostasis of essential trace elements including iron and copper plays a key role in the pathogenesis of a myriad of serious conditions including iron deficiency (ID) anemia, in which impaired cellular energy metabolism is prominent. Although experimental studies documented decreased activity of cytochrome c oxidase (CytOx) in ID, there are not enough clinical data. The present study was conducted to determine serum copper levels and activity of mitochondrial CytOx in isolated lymphocytes of patients with iron deficiency. MATERIAL AND METHODS: A total of 210 cases (2-17 years) were included in this prospective study. Serum iron and copper levels were measured. According to the serum iron levels, patients were allocated to iron deficient (ID, n = 70) and iron deficiency anemia (IDA, n = 70) groups, and iron-sufficient participants were allocated to the control group (n = 70). Activity of CytOx in the circulating lymphocytes was colorimetrically measured and compared with the controls. RESULTS: The CytOx activity was significantly higher in the IDA (2.9 ±1.2 mOD/min, n = 62) group compared to the control group (2.4 ±1.3 mOD/min, n = 68, p < 0.001). Interestingly, serum copper levels were significantly higher in both the ID (106.9 ±55.5 µg/dl, n = 64, p = 0.0001) and IDA (115.1 ±50.2 µg/dl, n = 59, p = 0.0001) groups than the control group (72.1 ±46.7 µg/dl, n = 69). CONCLUSIONS: Higher serum copper levels in patients with IDA implicate co-operative interaction between these trace elements. The elevated CytOx activity in patients with IDA is probably secondary to the normal/elevated serum copper levels.

Involvement of DFNB59 mutations in autosomal recessive nonsyndromic hearing impairment.

In a consanguineous Turkish family, a locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI) was mapped to chromosome 2q31.1-2q33.1. Microsatellite marker analysis in the complete family determined the critical linkage interval that overlapped with DFNB27, for which the causative gene has not yet been identified, and DFNB59, a recently described auditory neuropathy caused by missense mutations in the DFNB59 gene. The 352-amino acid (aa) DFNB59 gene product pejvakin is present in hair cells, supporting cells, spiral ganglion cells, and the first three relays of the afferent auditory pathway. A novel homozygous nonsense mutation (c.499C>T; p.R167X) was detected in the DFNB59 gene, segregating with the deafness in the family. The mRNA derived from the mutant allele was found not to be degraded in lymphocytes, indicating that a truncated pejvakin protein of 166 aa may be present in the affected individuals. Screening of 67 index patients from additional consanguineous Turkish families with autosomal recessive hearing impairment revealed a homozygous missense mutation (c.547C>T; p.R183W) that segregates with the hearing impairment in one family. Furthermore, in a panel of 83 Dutch patients, two additional novel mutations (c.509_512delCACT; p.S170CfsX35 and c.731T>G; p.L244R), which were not present in ethnically matched controls, were found heterozygously. Together, our data indicate that also nonsense mutations in DFNB59 cause nonsyndromic hearing loss, but that mutations in DFNB59 are not a major cause of nonsyndromic hearing impairment in the Turkish and Dutch population.