Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations.

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 µg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 µM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62 % of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labeled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and for the resulting neurotoxic effects.

The Multicenter Italian Trial Assesses the Performance of FDG-PET /CT Related to Pre-Test Cancer Risk in Patients with Solitary Pulmonary Nodules and Introduces a Segmental Thoracic Diagnostic Strategy.

PURPOSE: The Italian Tailored Assessment of Lung Indeterminate Accidental Nodule (ITALIAN) trial is a trial drawn to determine the performance of 18F-FDG-PET/CT in patients with solitary pulmonary nodules (SPN), stratified for a different kind of risk. An additional end-point was to compare the diagnostic information and estimated dosimetry, provided by a segmental PET/CT (s-PET/CT) acquisition instead of a whole body PET/CT (wb-PET/CT), in order to evaluate if segmental thoracic PET/CT can be used in patients with SPN. METHODS: 18F-FDG PET/CT of 502 patients, stratified for pre-test cancer risk, was retrospectively analyzed. FDG uptake in SPN was assessed by a 4-point scoring (4PS) system and a semiquantitative analysis using the ratio between SUVmax in SPN and SUVmean in mediastinal blood pool (BP), and between SUVmax in SPN and SUVmean in the liver (L). Histopathology and/or follow-up data were used as a standard of reference. Data obtained on the thoracic part of wb-PET/CT, defined as s - PET/CT, were compared with those deriving from wb-PET/CT. RESULTS: SPNs were malignant in 180 patients (36%), benign in 175 (35%), and indeterminate in 147 (29%). The 355 patients diagnosed with a definitive SPN nature (malignant or benign) were considered for the analysis of PET performance. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values, and accuracy were 85.6%, 85.7%, 86%, 85.2%, and 85.6%, respectively. Sensitivity and PPV were higher in intermediate and high-risk patients. 18F-FDG uptake indicative of thoracic and extra-thoracic lesions was detectable in 13% and 3% of the patients. Compared to wb-PET/CT, s-PET/CT could save about 2/3 of 18F-FDG dose, radiation exposure or scan-time, without affecting the clinical impact of PET/CT. CONCLUSION: In patients with SPN, the pre-test likelihood of malignancy stratification allows to better define PET clinical setting and its diagnostic power. In subjects with low-intermediate pre-test likelihood of malignancy, s-PET/CT might be planned in advance. The adoption of this segmental strategy could reduce radiation exposure, scan-time, and might allow individually targeted protocols.

Manganese Mapping Using a Fluorescent Mn2+ Sensor and Nanosynchrotron X-ray Fluorescence Reveals the Role of the Golgi Apparatus as a Manganese Storage Site.

Elucidating dynamics in transition-metal distribution and localization under physiological and pathophysiological conditions is central to our understanding of metal-ion regulation. In this Forum Article, we focus on manganese and specifically recent developments that point to the relevance of the Golgi apparatus in manganese detoxification when this essential metal ion is overaccumulated because of either environmental exposure or mutations in manganese efflux transporters. In order to further evaluate the role of the Golgi apparatus as a manganese-ion storage compartment under subcytotoxic manganese levels, we use a combination of confocal microscopy using a sensitive "turn-on" fluorescent manganese sensor, M1, and nanosynchrotron X-ray fluorescence imaging to show that manganese ions are stored in the Golgi apparatus under micromolar manganese exposure concentrations. Our results, along with previous reports on manganese accumulation, now indicate a central role of the Golgi apparatus in manganese storage and trafficking under subcytotoxic manganese levels and hint toward a possible role of the Golgi apparatus in manganese storage even under physiological conditions.

Gallium- and Iron-Pyoverdine Coordination Compounds Investigated by X-ray Photoelectron Spectroscopy and X-ray Absorption Spectroscopy.

Iron is an essential nutrient for nearly all forms of life, although scarcely available due to its poor solubility in nature and complex formation in higher eukaryotes. Microorganisms have evolved a vast array of strategies to acquire iron, the most common being the production of high-affinity iron chelators, termed siderophores. The opportunistic bacterial pathogen Pseudomonas aeruginosa synthesizes and secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), characterized by very different structural and functional properties. Due to its chemical similarity with Fe(III), Ga(III) interferes with several iron-dependent biological pathways. Both PVD and PCH bind Fe(III) and Ga(III). However, while the Ga-PCH complex is more effective than Ga(III) in inhibiting P. aeruginosa growth, PVD acts as a Ga(III) scavenger and protects bacteria from Ga(III) toxicity. To gain more insight into the different outcomes of the biological paths observed for the Fe(III) and Ga(III)-siderophore complexes, better knowledge is needed of their coordination geometries that directly influence the metal complexes chemical stability. The valence state and coordination geometry of the Ga-PCH and Fe-PCH complexes has recently been investigated in detail; as for PVD complexes, several NMR structural studies of Ga(III)-PVD are reported in the literature, using Ga(III) as a diamagnetic isosteric substitute for Fe(III). In this work, we applied up-to-date spectroscopic techniques as synchrotron-radiation-induced X-ray photoelectron spectroscopy (SR-XPS) and X-ray absorption fine structure (XAFS) spectroscopy coupled with molecular modeling to describe the electronic structure and coordination chemistry of Fe and Ga coordinative sites in PVD metal complexes. These techniques allowed us to unambiguously determine the oxidation state of the coordinative ions and to gather interesting information about the similarities and differences between the two coordination compounds as induced by the different metal.

SLC30A10 Mutation Involved in Parkinsonism Results in Manganese Accumulation within Nanovesicles of the Golgi Apparatus.

Manganese (Mn) is an essential metal that can be neurotoxic when elevated exposition occurs leading to parkinsonian-like syndromes. Mutations in the Slc30a10 gene have been identified in new forms of familial parkinsonism. SLC30A10 is a cell surface protein involved in the efflux of Mn and protects the cell against Mn toxicity. Disease-causing mutations block the efflux activity of SLC30A10, resulting in Mn accumulation. Determining the intracellular localization of Mn when disease-causing SLC30A10 mutants are expressed is essential to elucidate the mechanisms of Mn neurotoxicity. Here, using organelle fluorescence microscopy and synchrotron X-ray fluorescence (SXRF) imaging, we found that Mn accumulates in the Golgi apparatus of human cells transfected with the disease-causing SLC30A10-Δ105-107 mutant under physiological conditions and after exposure to Mn. In cells expressing the wild-type SLC30A10 protein, cellular Mn content was low after all exposure conditions, confirming efficient Mn efflux. In nontransfected cells that do not express endogenous SLC30A10 and in mock transfected cells, Mn was located in the Golgi apparatus, similarly to its distribution in cells expressing the mutant protein, confirming deficient Mn efflux. The newly developed SXRF cryogenic nanoimaging (<50 nm resolution) indicated that Mn was trapped in single vesicles within the Golgi apparatus. Our results confirm the role of SLC30A10 in Mn efflux and the accumulation of Mn in cells expressing the disease-causing SLC30A10-Δ105-107 mutation. Moreover, we identified suborganelle Golgi nanovesicles as the main compartment of Mn accumulation in SLC30A10 mutants, suggesting interactions with the vesicular trafficking machinery as a cause of the disease.

Uranium exposure of human dopaminergic cells results in low cytotoxicity, accumulation within sub-cytoplasmic regions, and down regulation of MAO-B.

Natural uranium is an ubiquitous element present in the environment and human exposure to low levels of uranium is unavoidable. Although the main target of acute uranium toxicity is the kidney, some concerns have been recently raised about neurological effects of chronic exposure to low levels of uranium. Only very few studies have addressed the molecular mechanisms of uranium neurotoxicity, indicating that the cholinergic and dopaminergic systems could be altered. The main objective of this study was to investigate the mechanisms of natural uranium toxicity, after 7-day continuous exposure, on terminally differentiated human SH-SY5Y cells exhibiting a dopaminergic phenotype. Cell viability was first assessed showing that uranium cytotoxicity only occurred at high exposure concentrations (> 125 µM), far from the expected values for uranium in the blood even after occupational exposure. SH-SY5Y differentiated cells were then continuously exposed to 1, 10, 125 or 250 µM of natural uranium for 7 days and uranium quantitative subcellular distribution was investigated by means of micro-PIXE (Particle Induced X-ray Emission). The subcellular element imaging revealed that uranium was located in defined perinuclear regions of the cytoplasm, suggesting its accumulation in organelles. Uranium was not detected in the nucleus of the differentiated cells. Quantitative analysis evidenced a very low intracellular uranium content at non-cytotoxic levels of exposure (1 and 10 µM). At higher levels of exposure (125 and 250 µM), when cytotoxic effects begin, a larger and disproportional intracellular accumulation of uranium was observed. Finally the expression of dopamine-related genes was quantified using real time qRT-PCR. The expression of monoamine oxidase B (MAO-B) gene was statistically significantly decreased after exposure to uranium while other dopamine-related genes were not modified. The down regulation of MAO-B was confirmed at the protein level. This original result suggests that the inhibition of dopamine catabolism, but also of other MAO-B substrates, could constitute selective effects of uranium neurotoxicity.

Synthesis and Structural Characterization of Silver Nanoparticles Stabilized with 3-Mercapto-1-Propansulfonate and 1-Thioglucose Mixed Thiols for Antibacterial Applications.

The synthesis, characterization and assessment of the antibacterial properties of hydrophilic silver nanoparticles (AgNPs) were investigated with the aim to probe their suitability for innovative applications in the field of nanobiotechnology. First, silver nanoparticles were synthetized and functionalized with two capping agents, namely 3-mercapto-1-propansulfonate (3MPS) and 1-ß-thio-d-glucose (TG). The investigation of the structural and electronic properties of the nano-systems was carried out by means of X-ray Photoelectron Spectroscopy (XPS) and X-ray Absorption Spectroscopy (XAS). XPS data provided information about the system stability and the interactions between the metallic surface and the organic ligands. In addition, XPS data allowed us to achieve a deep understanding of the influence of the thiols stoichiometric ratio on the electronic properties and stability of AgNPs. In order to shed light on the structural and electronic local properties at Ag atoms sites, XAS at Ag K-Edge was successfully applied; furthermore, the combination of Dynamic Light Scattering (DLS) and XAS results allowed determining AgNPs sizes, ranging between 3 and 13 nm. Finally, preliminary studies on the antibacterial properties of AgNPs showed promising results on four of six multidrug-resistant bacteria belonging to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.).

Bone mineral density in a population of children and adolescents with cerebral palsy and mental retardation with or without epilepsy.

PURPOSE: The present study aimed to assess bone mineral density (BMD) in a population of children and adolescents with cerebral palsy and mental retardation with or without epilepsy. METHODS: One hundred thirteen patients (63 male and 50 female) were recruited for evaluation. Patients were divided in three groups: 40 patients (group 1) were affected by cerebral palsy and mental retardation; 47 (group 2) by cerebral palsy, mental retardation, and epilepsy; and 26 (group 3) by epilepsy. The control group consisted of 63 healthy children and adolescents. Patients underwent a dual-energy x-ray absorptiometry (DEXA) scan of the lumbar spine (L1-L4), and z-score was calculated for each patient; t-score was considered for patients 18 years of age and older. KEY FINDINGS: Abnormal BMD by DEXA was found in 17 patients (42.5%) in group 1, in 33 (70.2%) in group 2, and in 3 (11.5%) in group 3. In groups 1 and 2, tetraparesis and severe/profound mental retardation were related to a significantly abnormal BMD (p = 0.003). The multivariate analysis of independent factors on BMD (z-score) revealed a significant correlation between BMD (z-score) and age (p = 0.04), body mass index (BMI; p = 0.002), severe/profound mental retardation (p = 0.03), and epilepsy (p = 0.05). SIGNIFICANCE: A significantly lower BMD z-score value was found in patients with cerebral palsy, mental retardation, and epilepsy compared with those without epilepsy. The epileptic disorder appears to be an aggravating factor on bone health when comorbid with cerebral palsy and mental retardation.

Incremental prognostic value of coronary flow reserve assessed with single-photon emission computed tomography.

BACKGROUND: We assessed the prognostic value of coronary flow reserve (CFR) estimated by single-photon emission computed tomography (SPECT) in patients with suspected myocardial ischemia. METHODS AND RESULTS: Myocardial perfusion and CFR were assessed in 106 patients using dipyridamole/rest Tc-99m sestamibi SPECT and follow-up was obtained in 103 (97%) patients. Four early revascularized patients were excluded and 99 were assigned to normal (summed stress score <3) vs abnormal myocardial perfusion and to normal (≥2.0) vs abnormal CFR. During the follow-up (5.8 ± 2.1 years), 28 patients experienced a cardiac event (cardiac death, nonfatal myocardial infarction, and late revascularization). Abnormal perfusion (P < .01) and abnormal CFR (P < .05) were independent predictors of cardiac events at Cox proportional hazard regression analysis. Also in patients with normal perfusion, abnormal CFR was associated with a higher annual event rate compared with normal CFR (5.2% vs 0.7%; P < .05). CFR data improved the prognostic power of the model including clinical and myocardial perfusion data increasing the global chi-square from 18.6 to 22.8 (P < .05). Finally, at parametric survival analysis, in patients with normal perfusion the time to achieve ≥2% risk of events was >60 months in those with normal and <12 months in those with abnormal CFR. CONCLUSIONS: Myocardial perfusion findings and CFR at SPECT imaging are both independent predictors of cardiac events. Estimated CFR provides incremental prognostic information over those obtained from clinical and myocardial perfusion data, particularly in patients with normal perfusion findings.

Zinc inhibits calcium-mediated and nitric oxide-mediated ion secretion in human enterocytes.

Zn(2+) is effective in the treatment of acute diarrhea, but its mechanisms are not completely understood. We previously demonstrated that Zn(2+) inhibits the secretory effect of cyclic adenosine monophosphate but not of cyclic guanosine monophosphate in human enterocytes. The aim of the present study was to investigate whether Zn(2+) inhibits intestinal ion secretion mediated by the Ca(2+) or nitric oxide pathways. To investigate ion transport we evaluated the effect of Zn(2+) (35 microM) on electrical parameters of human intestinal epithelial cell monolayers (Caco2 cells) mounted in Ussing chambers and exposed to ligands that selectively increased intracellular Ca(2+) (carbachol 10(-6)M) or nitric oxide (interferon-gamma 300 UI/ml) concentrations. We also measured intracellular Ca(2+) and nitric oxide concentrations. Zn(2+) significantly reduced ion secretion elicited by carbachol (-87%) or by interferon-gamma (-100%), and inhibited the increase of intracellular Ca(2+) and nitric oxide concentrations. These data indicate that Zn(2+) inhibits ion secretion elicited by Ca(2+) and nitric oxide by directly interacting with the enterocyte. They also suggest that Zn(2+) interferes with three of the four main intracellular pathways of intestinal ion secretion that are involved in acute diarrhea.

Zinc fights diarrhoea in HIV-1-infected children: in-vitro evidence to link clinical data and pathophysiological mechanism.

Diarrhoea-related morbidity is reduced by zinc supplementation in HIV-1-infected children. The mechanisms of this effect are largely undefined. We provide evidence for role for Tat (transactivating peptide produced by HIV-1) in the pathogenesis of diarrhoea in AIDS patients. In this study we showed that zinc, preventing Tat-induced fluid secretion, directly limits a specific mechanism of HIV-1-related diarrhoea. Our data support a 'zinc approach' in adjunct to specific antiretroviral therapy in HIV-1-infected children.

Growth hormone regulates intestinal ion transport through a modulation of the constitutive nitric oxide synthase-nitric oxide-cAMP pathway.

AIM: Growth hormone (GH) directly interacts with the enterocyte stimulating ion absorption and reducing ion secretion induced by agonists of cAMP. Since nitric oxide (NO) is involved in the regulation of transepithelial ion transport and acts as a second messenger for GH hemodynamic effects, we tested the hypothesis that NO may be involved in the resulting effects of GH on intestinal ion transport. METHODS: Electrical parameters reflecting trans-epithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers and exposed to GH and cholera toxin (CT) alone or in combination, in the presence or absence of the NO synthase (NOS) inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME). Similar experiments were conducted to determine cAMP and nitrite/nitrate concentrations. NOS expression was assayed by Western blot analysis. RESULTS: L-NAME causes total abrogation of absorptive and anti-secretory effects by GH on intestinal ion transport. In addition, L-NAME was able to inhibit the GH-effects on intracellular cAMP concentration under basal conditions and in response to CT. GH induced a Ca(2+)-dependent increase of nitrites/nitrates production, indicating the involvement of the constitutive rather than the inducible NOS isoform, which was directly confirmed by Western blot analysis. CONCLUSION: These results suggest that the GH effects on intestinal ion transport, either under basal conditions or in the presence of cAMP-stimulated ion secretion, are mediated at an intracellular level by the activity of cNOS.

Inhibitory effect of HIV-1 Tat protein on the sodium-D-glucose symporter of human intestinal epithelial cells.

OBJECTIVE: The pathophysiology of HIV-1-related intestinal dysfunction is largely unknown. We previously found that the transactivator factor peptide (Tat) produced by HIV-1 induces ion secretion and inhibits cell proliferation in human enterocytes. Because sugar malabsorption is a frequent feature in AIDS patients, we evaluated whether Tat inhibits intestinal glucose absorption. DESIGN AND METHODS: We measured Na-D-glucose symporter (SGLT-1) activity and determined its phenotypic expression in Caco-2 cells, in the presence and absence of Tat, in uptake experiments using a non-metabolized radiolabelled glucose analogue, and by western blot analysis, respectively. alpha-Tubulin staining was used to study the effects exerted by Tat on cell structure. RESULTS: Tat dose dependently inhibited glucose uptake by human enterocytes. This effect was prevented by anti-Tat polyclonal antibodies and by L-type Ca channels agonist Bay K8644. Western blot analysis of cellular lysates and brush-border membrane preparations showed that Tat induced SGLT-1 missorting. Tat also caused a dramatic decrease in alpha-tubulin staining, which indicates dysruption of the cytoskeleton organization. CONCLUSIONS: Tat acutely impairs intestinal glucose absorption through SGLT-1 missorting. This result indicates that Tat is directly involved in AIDS-associated intestinal dysfunction.

Zinc inhibits cholera toxin-induced, but not Escherichia coli heat-stable enterotoxin-induced, ion secretion in human enterocytes.

BACKGROUND: Because zinc deficiency in malnourished children is associated with severe diarrhea, use of zinc supplementation has been proposed as an adjunct to oral rehydration. However, the effects of zinc on enterocyte ion transport are largely unknown. The objective of the present study was to investigate the effects of zinc on transepithelial ion transport under basal conditions and under conditions of enterotoxin-induced ion secretion. METHODS: Ion transport was investigated by monitoring electrical parameters in human intestinal Caco-2 cells that were mounted in Ussing chambers and exposed to increasing concentrations of zinc, both in the absence and presence of either cholera toxin (CT) or Escherichia coli heat-stable enterotoxin (ST). Intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) concentrations were also determined. RESULTS: The addition of zinc to the luminal or basolateral side of enterocytes induced a chloride-dependent, dose-related decrease in short-circuit current, indicating ion absorption. It also resulted in a substantial reduction in CT-induced ion secretion and in cAMP concentration. E. coli ST-induced ion secretion and cGMP concentration were not affected. Ion absorption peaked at 35 mu mol/L zinc, whereas excess zinc load induced active ion secretion. CONCLUSIONS: By causing a decrease in cAMP concentration, zinc directly promotes ion absorption and substantially reduces CT-induced, but not E. coli ST-induced, ion secretion.