Ultrafine particulate matter pollution and dysfunction of endoplasmic reticulum Ca2+ store: A pathomechanism shared with amyotrophic lateral sclerosis motor neurons?

Increased risk of neurodegenerative diseases has been envisaged for air pollution exposure. On the other hand, environmental risk factors, including air pollution, have been suggested for Amyotrophic Lateral Sclerosis (ALS) pathomechanism. Therefore, the neurotoxicity of ultrafine particulate matter (PM0.1) (PM < 0.1 µm size) and its sub-20 nm nanoparticle fraction (NP20) has been investigated in motor neuronal-like cells and primary cortical neurons, mainly affected in ALS. The present data showed that PM0.1 and NP20 exposure induced endoplasmic reticulum (ER) stress, as occurred in cortex and spinal cord of ALS mice carrying G93A mutation in SOD1 gene. Furthermore, NSC-34 motor neuronal-like cells exposed to PM0.1 and NP20 shared the same proteomic profile on some apoptotic factors with motor neurons treated with the L-BMAA, a neurotoxin inducing Amyotrophic Lateral Sclerosis/Parkinson-Dementia Complex (ALS/PDC). Of note ER stress induced by PM0.1 and NP20 in motor neurons was associated to pathological changes in ER morphology and dramatic reduction of organellar Ca2+ level through the dysregulation of the Ca2+-pumps SERCA2 and SERCA3, the Ca2+-sensor STIM1, and the Ca2+-release channels RyR3 and IP3R3. Furthermore, the mechanism deputed to ER Ca2+ refilling (e.g. the so called store operated calcium entry-SOCE) and the relative currents ICRAC were also altered by PM0.1 and NP20 exposure. Additionally, these carbonaceous particles caused the exacerbation of L-BMAA-induced ER stress and Caspase-9 activation. In conclusion, this study shows that PM0.1 and NP20 induced the aberrant expression of ER proteins leading to dysmorphic ER, organellar Ca2+ dysfunction, ER stress and neurotoxicity, providing putative correlations with the neurodegenerative process occurring in ALS.

Size-Based Effects of Anthropogenic Ultrafine Particles on Lysosomal TRPML1 Channel and Autophagy in Motoneuron-like Cells.

Background: An emerging body of evidence indicates an association between anthropogenic particulate matter (PM) and neurodegeneration. Although the historical focus of PM toxicity has been on the cardiopulmonary system, ultrafine PM particles can also exert detrimental effects in the brain. However, only a few studies are available on the harmful interaction between PM and CNS and on the putative pathomechanisms. Methods: Ultrafine PM particles with a diameter < 0.1 µm (PM0.1) and nanoparticles < 20 nm (NP20) were sampled in a lab-scale combustion system. Their effect on cell tracking in the space was studied by time-lapse and high-content microscopy in NSC-34 motor neurons while pHrodo™ Green conjugates were used to detect PM endocytosis. Western blotting analysis was used to quantify protein expression of lysosomal channels (i.e., TRPML1 and TPC2) and autophagy markers. Current-clamp electrophysiology and Fura2-video imaging techniques were used to measure membrane potential, intracellular Ca2+ homeostasis and TRPML1 activity in NSC-34 cells exposed to PM0.1 and NP20. Results: NP20, but not PM0.1, reduced NSC-34 motor neuron movement in the space. Furthermore, NP20 was able to shift membrane potential of motor neurons toward more depolarizing values. PM0.1 and NP20 were able to enter into the cells by endocytosis and exerted mitochondrial toxicity with the consequent stimulation of ROS production. This latter event was sufficient to determine the hyperactivation of the lysosomal channel TRPML1. Consequently, both LC3-II and p62 protein expression increased after 48 h of exposure together with AMPK activation, suggesting an engulfment of autophagy. The antioxidant molecule Trolox restored TRPML1 function and autophagy. Conclusions: Restoring TRPML1 function by an antioxidant agent may be considered a protective mechanism able to reestablish autophagy flux in motor neurons exposed to nanoparticles.

Size-based effects of anthropogenic ultrafine particles on activation of human lung macrophages.

The anthropogenic particulate matter (PM), suspended air dust that can be inhaled by humans and deposited in the lungs, is one of the main pollutants in the industrialized cities atmosphere. Recent studies have shown that PM has adverse effects on respiratory diseases. These effects are mainly due to the ultrafine particles (PM0.1, PM < 100 nm), which, thanks to their PM size, are efficiently deposited in nasal, tracheobronchial, and alveolar regions. Pulmonary macrophages are a heterogeneous cell population distributed in different lung compartments, whose role in inflammatory response to injury is of particular relevance. In this study, we investigated the effect of PM0.1 on Human Lung Macrophages (HLMs) activation evaluated as proinflammatory cytokines and chemokine release, Reactive Oxygen Species (ROS) production and intracellular Ca2+concentration ([Ca2+]i). Furthermore, PM0.1, after removal of organic fraction, was fractionated in nanoparticles both smaller (NP20) and bigger (NP100) than 20 nm by a properlydeveloped analytical protocol, allowed isolating their individual contribution. Interestingly, while PM0.1 and NP20 induced stimulatory effects on HLM cytokines release, NP100 had not effect. In particular, PM0.1 induced IL-6, IL-1ß, TNF-α, but not CXCL8, release from HLMs. Moreover, PM0.1, NP20 and NP100 did not induce ß-glucuronidase release, a preformed mediator contained in HLMs. The long time necessary for cytokines release (18 h) suggested that PM0.1 and NP20 could induce ex-novo production of the tested mediators. Accordingly, after 6 h of incubation, PM0.1 and NP20 induced mRNA expression of IL-6, TNF-α and IL-1ß. Moreover, NP20 induced ROS production and [Ca2+]i increase in a time-dependent manner, without producing cytotoxicity. Collectively, the present data highlight the main proinflammatory role of NP20 among PM fractions. This is particularly of concern because this fraction is not currently covered by legal limits as it is not easily measured at the exhausts by the available technical methodologies, suggesting that it is mandatory to search for new monitoring techniques and strategies for limiting NP20 formation.

On-line fast analysis of light hydrocarbons, PAH and radicals by molecular-beam time of flight mass spectrometry.

Volatile organic compounds (VOC) and polycyclic aromatic hydrocarbons (PAH), emitted in the environment from a wide range of combustion sources, are hazardous to human health and considered important precursors of both primary and secondary particulate pollutants. In the present work, light hydrocarbons up to C9, as main components of combustion-derived VOC, and PAH produced in fuel-rich conditions of premixed ethylene flames were analyzed by implementing a molecular-beam time of flight mass spectrometer (MB-TOFMS), purposely built for on-line fast monitoring of the environmental impact of combustion systems. The reliability of the MB-TOFMS was preliminarily verified on a slightly-sooting flame, comparing the results with those obtained by batch sampling and gas chromatographic techniques. Electron ionization (EI) and multi-photon ionization (MPI) were used as MB-TOFMS sources and tested on combustion gases of a no-sooting premixed ethylene flame where VOC and PAH are present in traces not detectable with batch sampling and conventional analytical techniques. The mass identification accuracy was improved and guaranteed by systematically performing internal mass calibration, exploiting the formation of "in situ" clusters from combustion water in the molecular beam apparatus. Selective and sensitive monitoring of light hydrocarbons and PAH, derived from oxidation and pyrolysis reactions featuring combustion, was shown to be especially effective when using the MB-TOFMS equipped with MPI source. This technique showed to be effective also for the detection of radical species that are important for the risk assessment of aerosol and fundamental understanding of aerosol chemistry at a molecular level.

Blue and green luminescent carbon nanodots from controllable fuel-rich flame reactors.

The continuous synthesis in controlled gas flame reactors is here demonstrated as a very effective approach for the direct and easy production of structurally reproducible carbon nanodots. In this work, the design of a simple deposition system, inserted into the reactor, is introduced. A controlled flame reactor is employed in the present investigation. The system was optimized for the production of carbon nanoparticles including fluorescent nanocarbons. Blue and green fluorescent carbon could be easily separated from the carbon nanoparticles by extraction with organic solvents and characterized by advanced chemical (size exclusion chromatography and mass spectrometry) and spectroscopic analysis. The blue fluorescent carbon comprised a mixture of molecular fluorophores and aromatic domains; the green fluorescent carbon was composed of aromatic domains (10-20 aromatic condensed rings), bonded and/or turbostratically stacked together. The green-fluorescent carbon nanodots produced in the flame reactor were insoluble in water but soluble in N-methylpyrrolidinone and showed excitation-independent luminescence. These results provide insights for a simple and controlled synthesis of carbon nanodots with specific and versatile features, which is a promising pathway for their use in quite different applicative sectors of bioimaging.

Insights on Clusters Formation Mechanism by Time of Flight Mass Spectrometry. 1. The Case of Ethanol-Water Clusters.

In the present work, water clusters with the addition of an electrophilic molecule such as ethanol have been studied by time of flight mass spectrometry (TOFMS). Mass distributions of molecular clusters of ethanol, water, and ethanol-water mixed clusters were obtained by two different ionization methods: electron ionization (EI) and picosecond laser photo-ionization (PI) at a wavelength of 355 nm. It was shown that short pulse laser ionization increases the signal intensity and promotes the extension of the detected mass range of the clusters in comparison with EI. Much larger clusters were detected in our experiments with respect to the current literature. The autocorrelation function (AF) was introduced in the analysis of the composition of the water clusters in terms of fundamental periodicities for obtaining information on clusters formation mechanisms. Besides, it was found that ethanol molecules are capable of substitutional interaction with hydrogen-bonded water clusters in ethanol-water binary mixtures but the self-association of ethanol was the dominant process. Moreover, the increase of ethanol concentration promotes both the formation of hydrated ethanol clusters and the self-association of ethanol clusters in ethanol-water binary mixtures. The formation of water-rich clusters and subsequent metastable fragmentation were found to be the dominant processes determining the water-rich cluster distribution, irrespective of the ionization process, while the ionization process significantly affects the ethanol-rich cluster distribution.

Vibrationally resolved high-resolution NEXAFS and XPS spectra of phenanthrene and coronene.

We performed a combined experimental and theoretical study of the C1s Near-Edge X-ray Absorption Fine-Structure (NEXAFS) spectroscopy and X-ray Photoelectron Spectroscopy in the gas phase of two polycyclic aromatic hydrocarbons (phenanthrene and coronene), typically formed in combustion reactions. In the NEXAFS of both molecules, a double-peak structure appears in the C1s → LUMO region, which differ by less than 1 eV in transition energies. The vibronic coupling is found to play an important role in such systems. It leads to weakening of the lower-energy peak and strengthening of the higher-energy one because the 0 - n (n > 0) vibrational progressions of the lower-energy peak appear in nearly the same region of the higher-energy peak. Vibrationally resolved theoretical spectra computed within the Frank-Condon (FC) approximation and linear coupling model agree well with the high-resolution experimental results. We find that FC-active normal modes all correspond to in-plane vibrations.

Analysis of polycyclic aromatic hydrocarbon sequences in a premixed laminar flame by on-line time-of-flight mass spectrometry.

A time-of-flight mass spectrometer in reflectron configuration has been used for the real-time detection of combustion products. The products of a premixed laminar C2H4/O2 flame at atmospheric pressure were sampled along its axis, diluted with inert gas and carried to the ion source as a molecular beam under minimal perturbation. Electron ionization and different optical ionization sources are compared. Photoionization was achieved with laser radiation from a Nd:YAG nanosecond pulsed laser at two different wavelengths in the UV range (266 and 355 nm). The mass spectra obtained using laser wavelength of 355 nm and electron ionization present a series of ions regularly spaced by 18 m/z units up to m/z 2000. This series allowed precise calibration of the instrument for compounds of high molecular weight. Information on the chemical nature of the analyzed species has been obtained by comparing mass spectra produced with different ionization methods. In order to better understand the growth mechanisms, polycyclic aromatic hydrocarbon sequences have been analyzed by fast Fourier transform of the mass spectra.

Fluorescence spectroscopy of complex aromatic mixtures.

The contribution of two- to seven-ring polycyclic aromatic hydrocarbons (PAH) and of larger aromatic structures contained in complex PAH-laden mixtures collected in flames was evaluated by fluorescence spectroscopy. A composition procedure of the fluorescence spectra of individual PAHs, analyzed by gas chromatography/mass spectrometry (GC/MS) was applied for the evaluation of their contribution to the fluorescence spectra of PAH-laden mixtures. In this way, it was possible to put in evidence the contribution to the total fluorescence spectrum of high molecular weight aromatic species present in the PAH-laden mixtures and not detectable by GC/MS. Qualitative and quantitative interpretation of synchronous and conventional fluorescence spectra of PAH-laden mixtures formed in combustion processes was proposed. The composition procedure was showed to be reliable in the UV-visible region for samples dissolved in cyclohexane solutions, but failed in the UV region when the solvent contained heavy atoms, as in the case of dichloromethane. However, the heavy-atom solvent effect was not sufficient to explain the depression of the UV fluorescence signal. Energy transfer interaction between fluorene and other fluorescing PAHs was suggested to be also responsible for this effect on the basis of fluorescence studies performed on single PAHs and their mixtures in cyclohexane, methanol, and dichloromethane.

Oligomeric carbon and siloxane series observed by matrix-assisted laser desorption/ionisation and laser desorption/ionisation mass spectrometry during the analysis of soot formed in fuel-rich flames.

Oligomeric carbon and siloxane series have been observed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS), during the analysis of the dichloromethane (DCM)-soluble fractions of condensable material recovered from fuel-rich flames. Laser desorption (LD) spectra showed a pattern of oligomeric dimethyl-siloxane structures with a spacing of 74 u. The siloxane series appears to have originated as contamination of samples by silicone oil used to lubricate connections of polymer tubing. This was confirmed by extracting silicone tubing and silicone grease with DCM followed by MALDI-MS analysis. A series of peaks with a mass spacing of 24 u was also observed, superimposed on the continuum of unresolved organic ions. This oligomeric series appears to correspond to polycyclic aromatics separated by (mainly) ethylene bridges. Thus LD-MS appears to have revealed a series of soot precursors, intermediate between polycyclic aromatics and particulate soot, which was not detected by MALDI-MS. More detailed work is necessary to define these species with precision.