Cancer exosomes in urine: A promising biomarker source

In face of innovative therapeutic strategies for cancer diagnosis and treatment, and of the elusiveness of renal and urinary tract tumors, the identification of novel biomarkers for diagnosis, prognosis, selection and monitoring of therapies is a primary target of research. The concept of "precision medicine" for tailoring the oncologic management in a minimally invasive fashion is considered a new epoch in cancer management. Extracellular vesicles, and exosomes in particular, carry lipids, mRNAs, non-coding RNAs, DNA, and active proteins, are present in a variety of bodily fluids including urine. In fact, urinary exosomes contain most of the urinoma proteins. Exosomes are nanovesicles originating from an endocytic pathway of the endocellular membranes and upon release are actors of intercellular communication, able to induce phenotypic changes including tumorigenesis and metastasis, in recipient cells. Tumor-derived exosomes play critical roles in all stages of tumor development and metastasis of almost all cancer types. Exosomal proteins may serve as biomarkers for clinical applications, still to be validated. Please reword the next sentence. Not clear. Exosomes extracellular gather in urine, thus becoming a great resource for recovery of biomarkers and a promising non-invasive diagnostic instrument for renal disease. Liquid biopsies (body fluids) may be preferable to tumor tissue biopsies since they are less invasive. Urinary exosomes in particular are available in great quantity in a noninvasive way and are representative of each of the cells of the urinary tract. In the present review, we summarize our knowledge of the urine exosomes with a new vision as liquid biopsy and high-throughput techniques. The emerging metabolic signature of urinary exosomes is also discussed in terms of its potential clinical application

Exosomes as "translational" cancer promoter organelles

The term Exosome has come into use to define nanovesicles contained in multivesicular endosomes (MVE), secreted by fusion of MVE with the plasma membrane. Exosomes are secreted in vivo by almost any cell type and can be isolated from body fluids. Indeed, circulating vesicles account for both exosomes and microvesicles (MVs), which can be purified by various purification methods and fully discriminated according to their shape, size and CD markers. Due to their protein and RNA content,once internalized, exosomes have the potential to act as \u201ctranslational\u201d organelles, altering the expression pattern of recipient cells, their growth, and fate. In fact, exosomes are involved in many of physio-pathological processes, thereby including cancer

Beneficial effect of polyphenols in COVID-19 and the ectopic F1FO-ATP synthase: Is there a link?

COVID-19 has been proposed to be an endothelial disease, as endothelial damage and oxidative stress contribute to its systemic inflammatory and thrombotic events. Polyphenols, natural antioxidant compounds appear as promising agents to prevent and treat COVID-19. Polyphenols bind and inhibit the F1Fo-ATP synthase rotary catalysis. An early target of polyphenols may be the ectopic F1Fo-ATP synthase expressed on the endothelial plasma membrane. Among the pleiotropic beneficial action of polyphenols in COVID-19, modulation of the ecto-F1Fo-ATP synthase, lowering the oxidative stress produced by the electron transfer chain coupled to it, would not be negligible

Beneficial Effect of Antioxidants in Retinopathies: A New Hypothesis

The retina is the most oxygen consuming tissue of the body. Rod and cone photoreceptors efficiently carry out visual cascades, which are energetically costly processes. Data has recently been published that suggests that the metabolic support to phototransduction in the rod outer segment (OS) may originate directly in the OS, which is able to conduct aerobic metabolism. This oxygen-handling activity of the rod OS, which was never suspected before, appears to be a primary cause of the generation of reactive oxygen species directly inside the OS. Oxidative stress has been hypothesised to contribute to most of the neurodegenerative retinal pathologies, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa and photoreceptor cell death after retinal detachment. Many natural antioxidant compounds are routinely used in experimental or human therapies for preventing or delaying photoreceptor degeneration in those pathologies. Here it is proposed that the ultimate reason for the beneficial actions of antioxidants in preventing or retarding the effect on the retinal degenerative pathologies can be found in their action on reactive oxygen species generated by the ectopic mitochondrial electron transport chain (ETC) coupled to FoF1-ATP synthase in rod OS disks. In fact, if not adequately coupled, the ETC generates reactive oxygen species that, in turn, can act on the polyunsaturated fatty acids which the rod OS is rich in. If correct, the mechanism put forward here would provide a potential for the molecular basis of therapies with antioxidants for retinal degenerative diseases

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of pho-toreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is im-paired in several pathologies, including diabetes. In this study, we investigate whether hyperglyce-mic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) me-dium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phago-cytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells

An update of the chemiosmotic theory as suggested by possible proton currents inside the coupling membrane

Understanding how biological systems convert and store energy is a primary purpose of basic research. However, despite Mitchell's chemiosmotic theory, we are far from the complete description of basic processes such as oxidative phosphorylation (OXPHOS) and photosynthesis. After more than half a century, the chemiosmotic theory may need updating, thanks to the latest structural data on respiratory chain complexes. In particular, up-to date technologies, such as those using fluorescence indicators following proton displacements, have shown that proton translocation is lateral rather than transversal with respect to the coupling membrane. Furthermore, the definition of the physical species involved in the transfer (proton, hydroxonium ion or proton currents) is still an unresolved issue, even though the latest acquisitions support the idea that protonic currents, difficult to measure, are involved. Moreover, F o F 1 -ATP synthase ubiquitous motor enzyme has the peculiarity (unlike most enzymes) of affecting the thermodynamic equilibrium of ATP synthesis. It seems that the concept of diffusion of the proton charge expressed more than two centuries ago by Theodor von Grotthuss is to be taken into consideration to resolve these issues. All these uncertainties remind us that also in biology it is necessary to consider the Heisenberg indeterminacy principle, which sets limits to analytical questions

Modulation of the rod outer segment aerobic metabolism diminishes the production of radicals due to light absorption

Oxidative stress is a primary risk factor for both inflammatory and degenerative retinopathies. Our previous data on blue light-irradiated retinas demonstrated an oxidative stress higher in the rod outer segment (OS) than in the inner limb, leading to impairment of the rod OS extra-mitochondrial aerobic metabolism. Here the oxidative metabolism and Reactive Oxygen Intermediates (ROI) production was evaluated in purified bovine rod OS in function of exposure to different illumination conditions. A dose response was observed to varying light intensities and duration in terms of both ROI production and ATP synthesis. Pretreatment with resveratrol, inhibitor of F1Fo-ATP synthase, or metformin, inhibitor of the respiratory complex I, significantly diminished the ROI production. Metformin also diminished the rod OS Complex I activity and reduced the maximal OS response to light in ATP production. Data show for the first time the relationship existing in the rod OS between its -aerobic- metabolism, light absorption, and ROI production. A beneficial effect was exerted by metformin and resveratrol, in modulating the ROI production in the illuminated rod OS, suggestive of their beneficial action also in vivo. Data shed new light on preventative interventions for cone loss secondary to rod damage due to oxidative stress

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in bovine rod outer segments.

PURPOSE Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS

Continuous Glucose Monitoring in Preterm Infants: The Role of Nutritional Management in Minimizing Glycemic Variability

Glycemic variability (GV) is common in preterm infants. In the premature population, GV is a risk factor for morbidity and mortality. Both hypo- and hyperglycemia can impair neurodevelopment. We investigated the impact of continuous versus intermittent tube enteral feeding on GV. In our prospective observational study, 20 preterm infants with a gestational age ≤ 34 weeks at either continuous or intermittent bolus full enteral feeding. For five days, continuous glucose monitoring (CGM) was utilized, which was achieved through the subcutaneous insertion of a sensor. A total of 27,532 measurements of blood glucose were taken. The mean amplitude of glycemic excursions did not differ between the two cohorts statistically. Continuous feeding resulted in higher positive values, increasing the risk of hypo- and hyperglycemia. Subjects who were small for their gestational age had a higher standard deviation during continuous feeding (p = 0.001). Data suggest that intermittent bolus nutrition is better for glycemic control than continuous nutrition. Nutritional management optimization of preterm infants appears to be critical for long-term health. In the future, CGM may provide a better understanding of the optimal glucose targets for various clinical conditions, allowing for a more personalized approach to management

Biological surface properties in extracellular vesicles and their effect on cargo proteins

Ultracentrifugationon sucrose density gradientappears to be the best purification protocol for extracellular vesicle (EVs) purification. After this step, to reduce disulfide bridges linking exogenous proteins to the vesicles, the collected samples are routinely washed and treated with dithiothreitol (DTT). Such incubations are performed at temperatures ranging from room temperature up to 95\u2009\ub0C, with either Tris or PBS as buffers. We re-investigated these steps on both exosomes and microvesicles purified from blood (serum) and urine by electrophoretic separation, silver staining and western blots analysis. Data confirm that an extra centrifugation on a sucrose cushion can effectively eliminate contaminants. Tris buffer (50 Mm) and \u3b2-mercaptoethanol as a reducing agent at room temperature dramatically improved either sample cleaning. By contrast, especially for exosomes PBS buffer and DTT, above 37\u2009\ub0C, caused massive protein aggregations, yielding blurred SDS-PAGE gels in both samples. Immuno-blot analyses demonstrated that in PBS-DTT contamination with albumin (in serum) or with uromodulin (in urine) occurs. DTT, likely due to its two-SH groups, might form scrambled SS-bonds promoting EVs interaction with environmental macromolecules via disulphide bridges. Therefore, to obtain maximum vesicle purity for biomarker investigations and to maximize both presence of EVs proteins and their accessibility, use of DTT is not recommended