Proteomic Profiling of Cerebrospinal Fluid and Its Extracellular Vesicles from Extraventricular Drainage in Pediatric Pilocytic Astrocytoma, towards Precision Oncology.

Pediatric pilocytic astrocytoma (PA) is the most common brain tumor in children. Complete resection provides a favorable prognosis, except for unresectable PA forms. There is an incomplete understanding of the molecular and cellular pathogenesis of PA. Potential biomarkers for PA patients, especially the non-BRAF-mutated ones are needed. Cerebrospinal fluid (CSF) is a valuable source of brain tumor biomarkers. Extracellular vesicles (EVs), circulating in CSF, express valuable disease targets. These can be isolated from CSF from waste extraventricular drainage (EVD). We analyzed the proteome of EVD CSF from PA, congenital hydrocephalus (CH, non-tumor control), or medulloblastoma (MB, unrelated tumoral control) patients. A total of 3072 proteins were identified, 47.1%, 65.6%, and 86.2% of which were expressed in the unprocessed total and in its large-EV (LEV), and small-EV (SEV) fractions. Bioinformatics identified 50 statistically significant proteins in the comparison between PA and HC, and PA and MB patients, in the same fractions. Kinase enrichment analysis predicted five enriched kinases involved in signaling. Among these, only Cyclin-dependent kinase 2 (CDK2) kinase was overexpressed in PA samples. PLS-DA highlighted the inactive carboxypeptidase-like protein X2 (CPXM2) and aquaporin-4 (AQP4) as statistically significant in all the comparisons, with CPXM2 being overexpressed (validated by ELISA and Western blot) and AQP4 downregulated in PA. These proteins were considered the most promising potential biomarkers for discriminating among pilocytic astrocytoma and unrelated tumoral (MB) or non-tumoral conditions in all the fractions examined, and are proposed to be prospectively validated in the plasma for translational medicine applications.

Identification of Central Nervous System Oncologic Disease Biomarkers in EVs from Cerebrospinal Fluid (CSF) of Pediatric Patients: A Pilot Neuro-Proteomic Study.

Cerebrospinal fluid (CSF) is a biochemical-clinical window into the brain. Unfortunately, its wide dynamic range, low protein concentration, and small sample quantity significantly limit the possibility of using it routinely. Extraventricular drainage (EVD) of CSF allows us to solve quantitative problems and to study the biological role of extracellular vesicles (EVs). In this study, we implemented bioinformatic analysis of our previous data of EVD of CSF and its EVs obtained from congenital hydrocephalus with the aim of identifying a comprehensive list of potential tumor and non-tumor biomarkers of central nervous system diseases. Among all proteins identified, those enriched in EVs are associated with synapses, synaptosomes, and nervous system diseases including gliomas, embryonal tumors, and epilepsy. Among these EV-enriched proteins, given the broad consensus present in the recent scientific literature, we validated syntaxin-binding protein 1 (STXBP1) as a marker of malignancy in EVD of CSF and its EVs from patients with pilocytic astrocytoma and medulloblastoma. Our results show that STXBP1 is negatively enriched in EVs compared to non-tumor diseases and its downregulation correlates with adverse outcomes. Further experiments are needed to validate this and other EV markers in the blood of pediatric patients for translational medicine applications.

The Liaison between Metabolism and Oxidative Stress in Human Diseases.

Mitochondria have been the prerequisite to eukaryote complexity since their likely endosymbiotic origin, allowing a remarkable expansion in the number of genes expressed [...].

Cirsiliol and Quercetin Inhibit ATP Synthesis and Decrease the Energy Balance in Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Resistant Staphylococcus epidermidis (MRSE) Strains Isolated from Patients.

Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit F1Fo-ATP synthase (ATP synthase) and that bacteria need a constant energy production to maintain their homeostasis, we evaluated the effect of two flavones, cirsiliol (tri-hy-droxy-6,7-dimethoxyflavone) and quercetin (3,3,4,5,7-pentahydroxyflavone), on energy production and intracellular ATP content in a methicillin-resistant Staphylococcus aureus (MRSA) strain and a methicillin-resistant Staphylococcus epidermidis (MRSE) strain isolated from patients, comparing the results to those obtained by treating the bacteria with oligomycin, a specific ATP synthase Fo moiety inhibitor. Real-time quantitative ATP synthesis and total ATP content of permeabilized Gram-positive bacteria were assayed by luminometry. The results showed that cirsiliol and quercetin inhibited ATP synthase and decreased the intracellular ATP levels in both strains, although the effect was higher in MRSE. In addition, while cirsiliol and quercetin acted immediately after the treatment, oligomycin inhibited ATP synthesis only after 30 min of incubation, suggesting that the different responses may depend on the different permeability of the bacterial wall to the three molecules. Thus, cirsiliol and quercetin could be considered potential additions to antibiotics due to their ability to target ATP synthase, against which bacteria cannot develop resistance.

Crosstalk between the Rod Outer Segments and Retinal Pigmented Epithelium in the Generation of Oxidative Stress in an In Vitro Model.

Dysfunction of the retinal pigment epithelium (RPE) is associated with several diseases characterized by retinal degeneration, such as diabetic retinopathy (DR). However, it has recently been proposed that outer retinal neurons also participate in the damage triggering. Therefore, we have evaluated the possible crosstalk between RPE and photoreceptors in priming and maintaining oxidative damage of the RPE. For this purpose, we used ARPE-19 cells as a model of human RPE, grown in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) and unoxidized (UOx) or oxidized (Ox) mammalian retinal rod outer segments (OSs). ARPE-19 cells were efficient at phagocytizing rod OSs in both NG and HG settings. However, in HG, ARPE-19 cells treated with Ox-rod OSs accumulated MDA and lipofuscins and displayed altered LC3, GRP78, and caspase 8 expression compared to untreated and UOx-rod-OS-treated cells. Data suggest that early oxidative damage may originate from the photoreceptors and subsequently extend to the RPE, providing a new perspective to the idea that retinal degeneration depends solely on a redox alteration of the RPE.

Extracellular Vesicles as Source of Biomarkers in Glomerulonephritis.

Kidney disease is a global health and healthcare burden. Glomerulonephritis (Gn), both primary and secondary, is generally characterized by an inflammatory glomerular injury and may lead to end-stage renal disease. Kidney biopsy is fundamental to the diagnosis; however, kidney biopsy presents some concerns that may partly hamper the clinical process. Therefore, more accurate diagnostic tools are needed. Extracellular vesicles (EVs) are membranous vesicles released by cells and found in bodily fluids, including urine. EVs mediate intercellular signaling both in health and disease. EVs can have both harmful and cytoprotective effects in kidney diseases, especially Gn. Previous findings reported that the specific cargo of urinary EV contains an aerobic metabolic ability that may either restore the recipient cell metabolism or cause oxidative stress production. Here, we provide an overview of the most recent proteomic findings on the role of EVs in several aspects of glomerulopathies, with a focus on this metabolic and redox potential. Future studies may elucidate how the ability of EVs to interfere with aerobic metabolism and redox status can shed light on aspects of Gn etiology which have remained elusive so far.

Platelet Metabolic Flexibility: A Matter of Substrate and Location.

Platelets are cellular elements that are physiologically involved in hemostasis, inflammation, thrombotic events, and various human diseases. There is a link between the activation of platelets and their metabolism. Platelets possess considerable metabolic versatility. Although the role of platelets in hemostasis and inflammation is known, our current understanding of platelet metabolism in terms of substrate preference is limited. Platelet activation triggers an oxidative metabolism increase to sustain energy requirements better than aerobic glycolysis alone. In addition, platelets possess extra-mitochondrial oxidative phosphorylation, which could be one of the sources of chemical energy required for platelet activation. This review aims to provide an overview of flexible platelet metabolism, focusing on the role of metabolic compartmentalization in substrate preference, since the metabolic flexibility of stimulated platelets could depend on subcellular localization and functional timing. Thus, developing a detailed understanding of the link between platelet activation and metabolic changes is crucial for improving human health.

Analysis of Electric Field Stimulation in Blue Light Stressed 661W Cells.

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected to direct current electric field (EF) stimulation. Our findings revealed that EF stimulation induced protective effects in 661W cells from Li-induced stress by multiple defense mechanisms, such as increase in mitochondrial activity, gain in mitochondrial potential, increase in superoxide levels, and the activation of unfolded protein response (UPR) pathways, all leading to an enhanced cell viability and decreased DNA damage. Here, our genetic screen results revealed the UPR pathway to be a promising target to ameliorate Li-induced stress by EF stimulation. Thus, our study is important for a knowledgeable transfer of EF stimulation into clinical application.

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.

The Flavone Cirsiliol from Salvia x jamensis Binds the F1 Moiety of ATP Synthase, Modulating Free Radical Production.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.

Maximizing the Rod Outer Segment Yield in Retinas Extracted from Cattle Eyes.

The retina is a thin neuronal multilayer responsible for the detection of visual information. The first step in visual transduction occurs in the photoreceptor outer segment. The studies on photoreception and visual biochemistry have often utilized rod outer segments (OS) or OS disks purified from mammalian eyes. Literature reports several OS and disk purification procedures that rarely specify the procedure utilized to collect the retina from the eye. Some reports suggest the use of scissors, while others do not mention the issue as they declare to utilize frozen retinas. Because the OS are deeply embedded in the retinal pigmented epithelium (RPE), the detachment of the retina by a harsh pull-out can cause the fracture of the photoreceptor cilium. Here, we present a protocol maximizing OS yield. Eye semi-cups, obtained by hemisecting the eyeball and discarding the anterior chamber structures and the vitreous, are filled with Mammalian Ringer. After 10-15 min of incubation, the retinas spontaneously detach with their wealth of OS almost intact. The impressive ability of the present protocol to minimize the number of OS stuck inside the RPE, and therefore lost, compared with the classic procedure, is shown by confocal laser scanning microscopy analysis of samples stained ex vivo with a dye (MitoTracker deep red) that stains both retinal mitochondria and OS. Total protein assay of OS disks purified by either procedure also shows a 300% total protein yield improvement. The advantage of the protocol presented is its higher yield of photoreceptor OS for subsequent purification procedures, while maintaining the physiological features of the retina.

Weighted Gene Co-Expression Network Analysis and Support Vector Machine Learning in the Proteomic Profiling of Cerebrospinal Fluid from Extraventricular Drainage in Child Medulloblastoma.

Medulloblastoma (MB) is the most common pediatric malignant central nervous system tumor. Overall survival in MB depends on treatment tuning. There is aneed for biomarkers of residual disease and recurrence. We analyzed the proteome of waste cerebrospinal fluid (CSF) from extraventricular drainage (EVD) from six children bearing various subtypes of MB and six controls needing EVD insertion for unrelated causes. Samples included total CSF, microvesicles, exosomes, and proteins captured by combinatorial peptide ligand library (CPLL). Liquid chromatography-coupled tandem mass spectrometry proteomics identified 3560 proteins in CSF from control and MB patients, 2412 (67.7%) of which were overlapping, and 346 (9.7%) and 805 (22.6%) were exclusive. Multidimensional scaling analysis discriminated samples. The weighted gene co-expression network analysis (WGCNA) identified those modules functionally associated with the samples. A ranked core of 192 proteins allowed distinguishing between control and MB samples. Machine learning highlighted long-chain fatty acid transport protein 4 (SLC27A4) and laminin B-type (LMNB1) as proteins that maximized the discrimination between control and MB samples. Machine learning WGCNA and support vector machine learning were able to distinguish between MB versus non-tumor/hemorrhagic controls. The two potential protein biomarkers for the discrimination between control and MB may guide therapy and predict recurrences, improving the MB patients' quality of life.

Beneficial effect of polyphenols in COVID-19 and the ectopic F1 FO -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 F1 Fo -ATP synthase rotary catalysis. An early target of polyphenols may be the ectopic F1 Fo -ATP synthase expressed on the endothelial plasma membrane. Among the pleiotropic beneficial action of polyphenols in COVID-19, modulation of the ecto-F1 Fo -ATP synthase, lowering the oxidative stress produced by the electron transfer chain coupled to it, would not be negligible.

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 photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic 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) medium 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 phagocytosis 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.

Adenosine Blood Level: A Biomarker of White Matter Damage in Very Low Birth Weight Infants.

BACKGROUND: Very low birth weight infants are at risk of developing periventricular white matter lesions. We previously reported high blood adenosine levels in premature infants and infants with low birth weight. We asked whether blood adenosine levels could be related to the vulnerability of the maturing white matter to develop lesions. The present study aims at finding a biomarker for the early detection of brain white matter lesions that can profoundly influence the neurodevelopmental outcome, whose pathophysiology is still unclear. METHODS: Dried blood spots were prospectively collected for the newborn screening program and adenosine concentration measurements. Fifty-six newborns who tested four times for blood adenosine concentration (at days 3, 15, 30, and 40 post-birth) were included in the program. All infants underwent brain MRI at term equivalent age. Neurodevelopmental outcomes were studied with Griffiths Mental Development Scales (GMDS) at 12 ± 2 months corrected age. RESULTS: Blood adenosine concentration increased over time from a median of 0.75 µM at Day 3 to 1.46 µM at Day 40. Adenosine blood concentration >1.58 µM at Day 15 was significantly associated with brain white matter lesions at MRI (OR (95 % CI) of 50.0 (3.6-688.3), p-value < 0.001). A moderate negative correlation between adenosine at 15 days of life and GMDS at 12 ± 2 months corrected age was found. CONCLUSION: These findings suggest a potential role for blood adenosine concentration as a biomarker of creberal white matter lesions in very low birth weight infants.

Myelin sheath and cyanobacterial thylakoids as concentric multilamellar structures with similar bioenergetic properties.

There is a surprisingly high morphological similarity between multilamellar concentric thylakoids in cyanobacteria and the myelin sheath that wraps the nerve axons. Thylakoids are multilamellar structures, which express photosystems I and II, cytochromes and ATP synthase necessary for the light-dependent reaction of photosynthesis. Myelin is a multilamellar structure that surrounds many axons in the nervous system and has long been believed to act simply as an insulator. However, it has been shown that myelin has a trophic role, conveying nutrients to the axons and producing ATP through oxidative phosphorylation. Therefore, it is tempting to presume that both membranous structures, although distant in the evolution tree, share not only a morphological but also a functional similarity, acting in feeding ATP synthesized by the ATP synthase to the centre of the multilamellar structure. Therefore, both molecular structures may represent a convergent evolution of life on Earth to fulfill fundamentally similar functions.

Proteomic profiling of human amnion for preterm birth biomarker discovery.

Spontaneous preterm birth (PTB) complicates about 12% of pregnancies worldwide, remaining the main cause of neonatal morbidity and mortality. Spontaneous preterm birth PTBs is often caused by microbial-induced preterm labor, mediated by an inflammatory process threatening both maternal and newborn health. In search for novel predictive biomarkers of PTB and preterm prelabor rupture of the membranes (pPROM), and to improve understanding of infection related PTB, we performed an untargeted mass spectrometry discovery study on 51 bioptic mid zone amnion samples from premature babies. A total of 6352 proteins were identified. Bioinformatics analyses revealed a ranked core of 159 proteins maximizing the discrimination between the selected clinical stratification groups allowing to distinguish conditions of absent (FIR 0) from maximal Fetal Inflammatory Response (FIR 3) stratified in function of Maternal Inflammatory Response (MIR) grade. Matrix metallopeptidase-9 (MMP-9) was the top differentially expressed protein. Gene Ontology enrichment analysis of the core proteins showed significant changes in the biological pathways associated to inflammation and regulation of immune and infection response. Data suggest that the conditions determining PTB would be a transversal event, secondary to the maternal inflammatory response causing a breakdown in fetal-maternal tolerance, with fetal inflammation being more severe than maternal one. We also highlight matrix metallopeptidase-9 as a potential predictive biomarker of PTB that can be assayed in the maternal serum, for future investigation.

Proteomic profile of mesothelial exosomes isolated from peritoneal dialysis effluent of children with focal segmental glomerulosclerosis.

Peritoneal dialysis (PD) is the worldwide recognized preferred dialysis treatment for children affected by end-stage kidney disease (ESKD). However, due to the unphysiological composition of PD fluids, the peritoneal membrane (PM) of these patients may undergo structural and functional alterations, which may cause fibrosis. Several factors may accelerate this process and primary kidney disease may have a causative role. In particular, patients affected by steroid resistant primary focal segmental glomerulosclerosis, a rare glomerular disease leading to nephrotic syndrome and ESKD, seem more prone to develop peritoneal fibrosis. The mechanism causing this predisposition is still unrecognized. To better define this condition, we carried out, for the first time, a new comprehensive comparative proteomic mass spectrometry analysis of mesothelial exosomes from peritoneal dialysis effluent (PDE) of 6 pediatric patients with focal segmental glomerular sclerosis (FSGS) versus 6 patients affected by other primary renal diseases (No FSGS). Our omic study demonstrated that, despite the high overlap in the protein milieu between the two study groups, machine learning allowed to identify a core list of 40 proteins, with ANXA13 as most promising potential biomarker, to distinguish, in our patient population, peritoneal dialysis effluent exosomes of FSGS from No FSGS patients (with 100% accuracy). Additionally, the Weight Gene Co-expression Network Analysis algorithm identified 17 proteins, with PTP4A1 as the most statistically significant biomarker associated to PD vintage and decreased PM function. Altogether, our data suggest that mesothelial cells of FSGS patients are more prone to activate a pro-fibrotic machinery. The role of the proposed biomarkers in the PM pathology deserves further investigation. Our results need further investigations in a larger population to corroborate these findings and investigate a possible increased risk of PM loss of function or development of encapsulating peritoneal sclerosis in FSGS patients, thus to eventually carry out changes in PD treatment and management or implement new solutions.

Development of an Accurate Mass Retention Time Database for Untargeted Metabolomic Analysis and Its Application to Plasma and Urine Pediatric Samples.

Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.

The Hormetic Effect of Metformin: "Less Is More"?

Metformin (MTF) is the first-line therapy for type 2 diabetes (T2DM). The euglycemic effect of MTF is due to the inhibition of hepatic glucose production. Literature reports that the principal molecular mechanism of MTF is the activation of 5'-AMP-activated protein kinase (AMPK) due to the decrement of ATP intracellular content consequent to the inhibition of Complex I, although this effect is obtained only at millimolar concentrations. Conversely, micromolar MTF seems to activate the mitochondrial electron transport chain, increasing ATP production and limiting oxidative stress. This evidence sustains the idea that MTF exerts a hormetic effect based on its concentration in the target tissue. Therefore, in this review we describe the effects of MTF on T2DM on the principal target organs, such as liver, gut, adipose tissue, endothelium, heart, and skeletal muscle. In particular, data indicate that all organs, except the gut, accumulate MTF in the micromolar range when administered in therapeutic doses, unmasking molecular mechanisms that do not depend on Complex I inhibition.