Trajectories of trace element accumulation in seagrass (Posidonia oceanica) over a decade reveal the footprint of fish farming.

To evaluate the effect of trace element (TE) release from fish farms on seagrass Posidonia oceanica, we compared TE concentrations (As, Cd, Co, Cu, Mn, Mo, Ni, Pb, V, Zn) in shoots near fish cages (Station 'Cage') with those away from them (Station 'Control') in two fish farm facilities (Site 1 and Site 2, North Aegean Sea, Greece). We assessed the present (i.e., 2021, year of sampling) and past (reconstructed period 2012-2020) accumulation of TEs using the living compartments (leaf blades, sheaths, rhizomes, roots, epiphytes) and the dead sheaths, respectively. We also assessed possible seagrass degradation by reconstructing past rhizome production. P. oceanica rhizome production at the 'Cage' stations was up to 50% lower than at the 'Control' stations. Most TE concentrations were higher at 'Cage' stations, but the differences often depended on the seagrass living compartment. Significant differentiation between 'Cage' and 'Control' stations was observed based on the TE concentrations of the dead sheaths during 2012-2020. The contamination level at the 'Cage' stations was mostly moderate in Site 1 and low in Site 2, during the reconstructed period, while an increasing contamination trend was found for certain potential phytotoxic TEs (As, Cu, Cd, Mo, V). Our results emphasize the need for the aquaculture industry to work towards a more ecologically aware approach.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

Selenium, Selenoproteins and 10-year Cardiovascular Risk: Results from the ATTICA Study.

BACKGROUND: Selenium (Se) is an essential trace element that is involved in several pathophysiological functions. The relationship of Se with cardiovascular disease remains inconclusive, especially regarding the role of different selenospecies. OBJECTIVE: The present study assessed the levels of Se distribution in plasma selenoproteins, namely glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and selenoalbumin (SeAlb) and total Se in selenoproteins in relation to 10-year cardiovascular risk in the ATTICA prospective study. METHODS: A sub-sample from the ATTICA Study's database, consisting of 278 subjects (114 women and 164 men) with data on Se and selenoproteins levels, was considered. SeGPx3, SelP, and SeAlb in human plasma were simultaneously determined by high-performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) at baseline. The duration of the follow-up was 8.74 ±2.36 years (mean± standard deviation) and cardiovascular outcomes were recorded. Cox proportional hazards models were applied with total Se or selenoprotein Se as independent variables adjusted for several covariates. RESULTS: Total Se in selenoproteins was positively related to 10-year relative risk of cardiovascular disease (Hazard Ratios of 3rd vs 2nd tertile 10.02, 95% CI:1.15, 92.34). Subjects with high Se but low SeGPx3, as identified by discordant percentiles in the distribution of SeGPx3 and Se, had a higher cardiovascular risk. CONCLUSION: The differentiated effects of circulating selenoproteins on cardiovascular disease risk in the present study, suggest the importance of redox regulation by specific selenoproteins.

Chlorfenapyr metabolism by mosquito P450s associated with pyrethroid resistance identifies potential activation markers.

Chlorfenapyr is a pro-insecticide increasingly used in combination with pyrethroids such as a-cypermethrin or deltamethrin in insecticide treated bednets (ITNs) to control malaria transmitted by pyrethroid-resistant mosquito populations. Chlorfenapyr requires P450 activation to produce tralopyril and other bioactive metabolites. Pyrethroid resistance is often associated with elevated levels of chemoprotective P450s with broad substrate specificity, which could influence chlorfenapyr activity. Here, we have investigated chlorfenapyr metabolism by a panel of eight P450s commonly associated with pyrethroid resistance in An. gambiae and Ae. aegypti, the major vectors of malaria and arboviruses. Chlorfenapyr was activated to tralopyril by An. gambiae CYP6P3, CYP9J5, CYP9K1 and Ae. aegypti, CYP9J32. The Kcat/KM value of 0.66 µM-1 min-1 for CYP9K1 was, 6.7 fold higher than CYP6P3 and CYP9J32 (both 0.1 µM-1 min-1) and 22-fold higher than CYP9J5 (0.03 µM-1 min-1). Further investigation of the effect of -cypermethrin equivalent to the ratios used with chlorfenapyr in bed nets (~ 1:2 molar ratio) resulted in a reduction in chlorfenapyr metabolism by CYP6P3 and CYP6K1 of 76.8% and 56.8% respectively. This research provides valuable insights into the metabolism of chlorfenapyr by mosquito P450s and highlights the need for continued investigation into effective vector control strategies.

Investigating the behavior of ultratrace levels of nanoparticulate and ionic silver in a seawater mesocosm using single particle inductively coupled plasma - mass spectrometry.

Silver nanoparticles (AgNPs) nowadays appear in close to 24% of consumer products that contain engineered nanomaterials. Thus, they are expected to be released into the environment, where their fate and effect are still undetermined. Considering the evidenced efficacy of the single particle Inductively Coupled Plasma - Mass Spectrometry (sp ICP-MS) technique in the study of nanomaterials, this work reports on the use of sp ICP-MS along with an online dilution sample introduction system for the direct analysis of untreated and spiked seawater samples, as part of a larger scale experiment studying the fate of Ag (ionic and nanoparticles) in seawater mesocosm systems. Silver nanoparticles coated with branched polyethyleneimine (BPEI@AgNPs) or ionic silver (Ag+) were introduced gradually into the seawater mesocosm tanks at very low, environmentally relevant concentrations (50 ng Ag L-1 per day, for 10 consecutive days, up to a total of 500 ng Ag L-1), and samples were collected and analyzed daily, within a consistent time window. Using very low detector dwell time (75 µs) and specialized data treatment, information was obtained on the nanoparticles' size distribution and particle number concentration, as well as the ionic silver content, of both the AgNPs and the Ag+ treated seawater mesocosm tanks. The results for the AgNP treated samples indicated the rapid degradation of the added silver particles, and the subsequent increase of ionic silver, with recoveries close to 100% for the first days of the experiment. On the other hand, particle formation was observed in the Ag+ treated seawater tanks, and even though the number concentration of silver-containing nanoparticles increased throughout the experiment, the amount of silver per particle remained relatively constant from the early days of the experiment. In addition, the online dilution sample introduction system for the ICP-MS proved capable of handling the untreated seawater matrix without significant contamination issues and downtime, while the low dwell time and data treatment procedure developed were shown to be suitable for the analysis of nanomaterials at the low nm-scale, despite the complex and heavy matrix introduced into the ICP-MS.

The Molecular Context of Oxidant Stress Response in Cancer Establishes ALDH1A1 as a Critical Target: What This Means for Acute Myeloid Leukemia.

The protein family of aldehyde dehydrogenases (ALDH) encompasses nineteen members. The ALDH1 subfamily consists of enzymes with similar activity, having the capacity to neutralize lipid peroxidation products and to generate retinoic acid; however, only ALDH1A1 emerges as a significant risk factor in acute myeloid leukemia. Not only is the gene ALDH1A1 on average significantly overexpressed in the poor prognosis group at the RNA level, but its protein product, ALDH1A1 protects acute myeloid leukemia cells from lipid peroxidation byproducts. This capacity to protect cells can be ascribed to the stability of the enzyme under conditions of oxidant stress. The capacity to protect cells is evident both in vitro, as well as in mouse xenografts of those cells, shielding cells effectively from a number of potent antineoplastic agents. However, the role of ALDH1A1 in acute myeloid leukemia has been unclear in the past due to evidence that normal cells often have higher aldehyde dehydrogenase activity than leukemic cells. This being true, ALDH1A1 RNA expression is significantly associated with poor prognosis. It is hence imperative that ALDH1A1 is methodically targeted, particularly for the acute myeloid leukemia patients of the poor prognosis risk group that overexpress ALDH1A1 RNA.

Increased metabolism in combination with the novel cytochrome b target-site mutation L258F confers cross-resistance between the Qo inhibitors acequinocyl and bifenazate in Tetranychus urticae.

Acequinocyl and bifenazate are potent acaricides acting at the Qo site of complex III of the electron transport chain, but frequent applications of these acaricides have led to the development of resistance in spider mites. Target-site resistance caused by mutations in the conserved cd1- and ef-helices of the Qo pocket of cytochrome b has been elucidated as the main resistance mechanism. We therefore monitored Qo pocket mutations in European field populations of Tetranychus urticae and uncovered a new mutation, L258F. The role of this mutation was validated by revealing patterns of maternal inheritance and by the independently replicated introgression in an unrelated susceptible genetic background. However, the parental strain exhibited higher resistance levels than conferred by the mutation alone in isogenic lines, especially for acequinocyl, implying the involvement of strong additional resistance mechanisms. This was confirmed by revealing a polygenic inheritance pattern with classical genetic crosses and via synergism experiments. Therefore, a genome-wide expression analysis was conducted that identified a number of highly overexpressed detoxification genes, including many P450s. Functional expression revealed that the P450 CYP392A11 can metabolize bifenazate by hydroxylation of the ring structure. In conclusion, the novel cytochrome b target-site mutation L258F was uncovered in a recently collected field strain and its role in acequinocyl and bifenazate resistance was validated. However, the high level of resistance in this strain is most likely caused by a combination of target-site resistance and P450-based increased detoxification, potentially acting in synergism.

A Triphenylphosphonium-Functionalized Delivery System for an ATM Kinase Inhibitor That Ameliorates Doxorubicin Resistance in Breast Carcinoma Mammospheres.

The enzyme ataxia-telangiectasia mutated (ATM) kinase is a pluripotent signaling mediator which activates cellular responses to genotoxic and metabolic stress. It has been shown that ATM enables the growth of mammalian adenocarcinoma stem cells, and therefore the potential benefits in cancer chemotherapy of a number of ATM inhibitors, such as KU-55933 (KU), are currently being investigated. We assayed the effects of utilizing a triphenylphosphonium-functionalized nanocarrier delivery system for KU on breast cancer cells grown either as a monolayer or in three-dimensional mammospheres. We observed that the encapsulated KU was effective against chemotherapy-resistant mammospheres of breast cancer cells, while having comparably lower cytotoxicity against adherent cells grown as monolayers. We also noted that the encapsulated KU sensitized the mammospheres to the anthracycline drug doxorubicin significantly, while having only a weak effect on adherent breast cancer cells. Our results suggest that triphenylphosphonium-functionalized drug delivery systems that contain encapsulated KU, or compounds with a similar impact, are a useful addition to chemotherapeutic treatment schemes that target proliferating cancers.

Innate Immune Responses to RSV Infection Facilitated by OGG1, an Enzyme Repairing Oxidatively Modified DNA Base Lesions.

The primary cause of morbidity and mortality from infection with respiratory syncytial virus (RSV) is the excessive innate immune response(s) (IIR) in which reactive oxygen species (ROS) play key role(s). However, the mechanisms for these processes are not fully understood. We hypothesized that expressions of IIR genes are controlled by the ROS-generated epigenetic-like mark 7,8-dihydro-8-oxo(d)guanine (8-oxo(d)Gua) and 8-oxoguanine DNA glycosylase1 (OGG1). Here, we report that ROS not only generates intrahelical 8-oxo(d)Gua, but also enzymatically disables OGG1 in RSV-infected human airway epithelial cells and mouse lungs. OGG1 bound to 8-oxo(d)Gua in gene regulatory sequences promotes expression of IIR genes, and consequently exacerbates lung inflammation, histological changes, and body weight loss of experimental animals. Pharmacological inhibition of OGG1 substrate binding decreased expression of RSV-induced chemokine and cytokines and significantly lessened clinical symptoms. Results of mechanistic studies show that OGG1 binding at 8-oxo(d)Gua promoter regions modulated loading of transcription factors via transient cooperative interactions in RSV-infected lungs and airway epithelial cells. Other base specific DNA repair proteins had no effects. Collectively, this study identifies unprecedented roles of ROS-generated DNA base lesion(s) and cognate repair protein as a determinant of RSV-induced exuberant inflammation. Pharmaceutical inhibition of OGG1 interaction with its DNA substrate may represent a novel strategy in prevention/intervention of respiratory viral infections.

Gold nanoclusters as elemental label for the sequential quantification of apolipoprotein E and metallothionein 2A in individual human cells of the retinal pigment epithelium using single cell-ICP-MS.

Gold nanoclusters (AuNCs) with a diameter of 1.99 nm on average were synthesized and applied as labels in immunoprobes for the determination of cytosolic proteins in individual human retinal pigment epithelium (HRPEsv) cells by single cell - inductively coupled plasma - mass spectrometry (sc-ICP-MS). For quantitative purposes, the number of gold atoms per immunoprobe (i.e., the amplification factor) was determined; 466 gold atoms on average were obtained. Human metallothioneins (MT), including the 2A isoform (MT2A), and apolipoprotein E (APOE) play an important role under inflammation and oxidation processes in the RPE. The new single biomarker strategy introduced was applied to the sequential determination of MT2A and APOE in HRPEsv cells under pro-inflammatory and control conditions through the development of immunoassays with the corresponding AuNCs immunoprobes and the measurement of the 197Au+ signal by sc-ICP-MS. In addition, 56Fe+ signal was measured as constituent element of HRPEsv cells in order to check the integrity of the cells after the immunoassay and to confirm the number of cell events detected when monitoring the protein label (197Au+). Optimisation of parameters related with the sample preparation for the analysis of cytosolic proteins in intact HRPEsv cells was carried out. The method was successfully applied to the determination of both proteins in control cells and cells treated with the recombinant human interleukin-1α. Quantitative results obtained per cell for the average protein amounts of APOE and MT2A using the sc-ICP-MS procedure were corroborated with commercial ELISA kits.

Metals in tissues of marine fish from the Thermaikos Gulf, Eastern Mediterranean Sea: Detection of changes with trophic level.

Concentrations of 27 metals and elements were investigated in the tissues of 28 demersal and pelagic marine fish species from the Eastern Mediterranean Sea. The analysis indicated highest concentrations of accumulation for the majority of elements in the gills, liver, and scales; whereas, lowest assimilation of elements was reported in the muscle. Pelagic fish accumulated higher concentrations of elements than demersal fish. An inverse correlation due to the growth dilution effect was revealed among element concentrations in the gills, liver, muscle, and skin tissues, and fish size. Patterns of biomagnification for Cs solely were recorded in the muscle and patterns of biodilution for most elements were evident in the gills, liver, muscle, and skin tissues. A positive correlation was determined between trophic level and element concentrations in the scales which could not only be associated with fish diet, but also with metal concentrations in the ambient environment and scales size.

Chip-based microfluidics on-line with inductively coupled plasma - mass spectrometry for standard dilution analysis.

Microfluidics coupled on-line with ICP-MS detection can be combined with powerful quantitation procedures that take advantage of internal standardization and standard additions, such as the recently introduced Standard Dilution Analysis (SDA). Although so far used at mL min-1 flow rates, here we demonstrate that SDA can be conveniently employed with a microfluidic chip-based ICP-MS system to improve determination accuracy for various sample types, including water, biological and cell digest samples, analyzed at µL min-1 flow rates. The efficient coupling of a microfluidic chip to ICP-MS was accomplished using a combination of commercially available components, including a pneumatic high-efficiency nebulizer and a spray chamber designed to allow for the addition of a laminar flow makeup gas. The addition of the makeup gas was crucial in order to avoid detrimental suction effects that can disrupt the operation of the microfluidic chip and cause signal instability, while it still allowed for the highly sensitive detection of metal isotopes by using ICP-MS. All mixing and dilution operations of the sample with the two calibration solutions required for SDA were performed in an automated and highly reproducible fashion on the microfluidic chip with the assistance of an external distributor valve. High average recoveries (97.4-100.1%) and low average relative standard deviations (2.9-4.8%) were achieved for the determined elements (Cd, Co, Pb, Cr) across several spiked matrices and certified reference materials, whereas only 140 µL of sample is required for SDA in triplicate or 40 µL for a single analysis. Hence, accuracy, precision, limited sample consumption, and the elimination of the need for manual sample dilution and mixing manipulations are some of the advantages of this newly developed chip-based microfluidic SDA ICP-MS technique.

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest.

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR.

Geographical origin and botanical type honey authentication through elemental metabolomics via chemometrics.

The trace and rare earth elements content of 93 honeys of different botanical type and origin have been studied through ICP-MS. Discriminant Analysis (DA) was successful for botanical type and geographical origin classification while Cluster Analysis (CA) was successful only for botanical type. Through Probabilistic Neural Network (PNN) analysis, 85.3% were correctly classified by the network according to their geographical origin and 73.3% according to their organic characterization. A Partial Least Squares (PLS) model was constructed, giving a prediction accuracy of more than 95%. Information obtained using Rare Earths (Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) and trace elements (Li, Mg, Mn, Ni, Co, Cu, Sr, Ba, Pb) via chemometric evaluation facilitated classification of honey samples.

Fish farming, metals and antibiotics in the eastern Mediterranean Sea: Is there a threat to sediment wildlife?

Chemical residues released from aquaculture farms may persist in the environment and may pose adverse ecological effects. The aim of this study was to assess the pollution status of marine sediments underneath or close to aquaculture farms in the Eastern Mediterranean Sea, the factors that affect the elemental distribution, and the exceedance of environmental quality standards and factors. To this end, surface sediment samples were collected (underneath fish cages, at 25 m distance and from a reference station) from 48 fish farms in Greece with variable environmental and geochemical characteristics. The content of 29 metals and major and trace elements, and three antibiotics (oxytetracycline, florfenicol, and flumequine), was determined in the sediment samples. Most of the elements found in the sediments originated from geological sources and their concentrations were close to Earth's Crust content. Below and close to fish farm cages, the sediment was enriched with P, Cu, Zn, Mo, and Cd mainly due to the aquaculture biosolid deposition, and changes in environmental conditions (increased organic matter, low redox potential). Cr and As were found to exceed twice the upper threshold limit of the available sediment quality guidelines in 13.5% and 7.3% of sampling stations, respectively. The potential ecological risk of V, Cr, Mn, Co, Ni, Cu, Zn, As and Pb was found to be low in the sediment close to fish cages except for Cd, which may pose a moderate to considerable risk in 12.5% of sampled fish farms. However, the estimated risks for this metal may be influenced by the high background levels found in the investigated areas. This study also shows that the occurrence of antibiotics in sediments below fish farms in the Eastern Mediterranean Sea is very sparse (i.e., only flumequine was found in two farms) and concentration levels are relatively low.

Versatile Dual-Inlet Sample Introduction System for Multi-Mode Single Particle Inductively Coupled Plasma Mass Spectrometry Analysis and Validation.

Metal-containing nanoparticles (NP) can be characterized with inductively coupled plasma mass spectrometers (ICP-MS) in terms of their size and number concentration by using the single-particle mode of the instrument (spICP-MS). The accuracy of measurement depends on the setup, operational conditions of the instrument and specific parameters that are set by the user. The transport efficiency of the ICP-MS is crucial for the quantification of the NP and usually requires a reference material with homogenous size distribution and a known particle number concentration. Currently, NP reference materials are available for only a few metals and in limited sizes. If particles are characterized without a reference standard, the results of both size and particle number may be biased. Therefore, a dual-inlet setup for characterizing nanoparticles with spICP-MS was developed to overcome this problem. This setup is based on a conventional introduction system consisting of a pneumatic nebulizer (PN) for nanoparticle solutions and a microdroplet generator (µDG) for ionic calibration solutions. A new and flexible interface was developed to facilitate the coupling of µDG, PN and the ICP-MS system. The interface consists of available laboratory components and allows for the calibration, nanoparticle (NP) characterization and cleaning of the arrangement, while the ICP-MS instrument is still running. Three independent analysis modes are available for determining particle size and number concentration. Each mode is based on a different calibration principle. While mode I (counting) and mode III (µDG) are known from the literature, mode II (sensitivity), is used to determine the transport efficiency by inorganic ionic standard solutions only. It is independent of NP reference materials. The µDG based inlet system described here guarantees superior analyte sensitivities and, therefore, lower detection limits (LOD). The size dependent LODs achieved are less than 15 nm for all NP (Au, Ag, CeO2) investigated.

Targeting breast cancer stem-like cells using chloroquine encapsulated by a triphenylphosphonium-functionalized hyperbranched polymer.

Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor progression and drug resistance. Accumulating evidence provides insight into the function of autophagy in maintenance and survival of CSCs. Here, we studied the impact of a mitochondriotropic triphenylphosphonium-functionalized dendrimeric nanocarrier on cultured breast cancer cell lines, grown either as adherent cells or as mammospheres that mimic a stem-like phenotype. The nanocarrier manifested a substantial cytotoxicity both alone as well as after encapsulation of chloroquine, a well-known autophagy inhibitor. The cytotoxic effects of the nanocarrier could be ascribed to interference with mitochondrial function. Importantly, mammospheres were selectively sensitive to encapsulated chloroquine and this depends on the expression of the gene encoding ATM kinase. Ataxia-telangiectasia mutated (ATM) kinase is an enzyme that functions as an essential signaling mediator that enables growth of cancer stem cells through the regulation of autophagy. We noted that this ATM-dependent sensitivity of mammospheres to encapsulated chloroquine was independent of the status of the tumor suppressor gene p53. Our study suggests that breast cancer stem cells, as they are modeled by mammospheres, are sensitive to encapsulated chloroquine, depending on the expression of the ATM kinase, which is thereby characterized as a potential biomarker for sensitivity to this type of treatment.

Biodegradation of phenol by Chlamydomonas reinhardtii.

The data presented in this particular study demonstrate that the biodegradation of phenol by Chlamydomonas reinhardtii is a dynamic bioenergetic process mainly affected by the production of catechol and the presence of a growth-promoting substrate in the culture medium. The study focused on the regulation of the bioenergetic equilibrium resulting from production of catechol after phenol oxidation. Catechol was identified by HPLC-UV and HPLC-ESI-MS/MS. Growth measurements revealed that phenol is a growth-limiting substrate for microalgal cultures. The Chlamydomonas cells proceed to phenol biodegradation because they require carbon reserves for maintenance of homeostasis. In the presence of acetic acid (a growth-promoting carbon source), the amount of catechol detected in the culture medium was negligible; apparently, acetic acid provides microalgae with sufficient energy reserves to further biodegrade catechol. It has been shown that when microalgae do not have sufficient energy reserves, a significant amount of catechol is released into the culture medium. Chlamydomonas reinhardtii acts as a versatile bioenergetic machine by regulating its metabolism under each particular set of growth conditions, in order to achieve an optimal balance between growth, homeostasis maintenance and biodegradation of phenol. The novel findings of this study reveal a paradigm showing how microalgal metabolic versatility can be used in the bioremediation of the environment and in potential large-scale applications.

Improved validation for single particle ICP-MS analysis using a pneumatic nebulizer / microdroplet generator sample introduction system for multi-mode nanoparticle determination.

This study reports on the development of a single-particle (sp) inductively coupled plasma mass spectrometry (ICP-MS) technique suitable for the multi-mode determination of nanoparticle (NP) metal mass fraction and number concentration. The described technique, which is based on a dual inlet system consisting of a pneumatic nebulizer (PN) and a microdroplet generator (MDG), allows for the sequential introduction of ionic metal calibrant solutions and nanoparticle suspensions via all combinations of the two inlets; thus allowing for a combination of three independent modes of analysis. A novel interface, assembled using standard analytical components (a demountable quartz ICP-MS torch, flexible non-conducting silicon tubing and various connectors), was used to interface the dual inlet system to an ICP-MS. The interface provided improved functionality, compared to a previous design. It is now possible to conveniently exchange and introduce standard solutions and samples via all inlet combinations, analyze them, and also wash the sample inlet systems while the whole setup is still connected to an operating ICP-MS. This setup provided seamless and robust operation in a total of three analysis modes, i.e. three ways to independently determine the metal mass fraction and NP number concentration. All three analyses modes could be carried out within a single analytical run lasting approximately 20 min. The unique feature of the described approach is that each analysis mode is based on a different calibration principle, thus constituting an independent way to determine metal mass fractions and nanoparticle number concentrations. Conducting the three independent state-of-the-art analysis, within a single analytical run, improves substantially the validation capabilities of sp-ICP-MS for NP analysis. To assess the technique's analytical performance, Au, Ag and CeO2 nanoparticles were analyzed. The determined average diameters for Au (56.7 ± 1.5 nm), Ag (72.8 ± 3.4 nm) and CeO2 (69.0 ± 6.4 nm) NPs were in close agreement for all three modes of analysis, as well as with the values provided by suppliers' for Au and Ag NPs (56.0 ± 0.5 for Au, 74.6 ± 3.8 nm for Ag). However, the determined average value for CeO2 was much higher than the expected 28.4 ± 10.4 nm, possibly due to NP agglomeration and the inability to detect NPs existing within the lower size range. The determined NP number concentrations, using analysis modes -I and -II, gave recoveries between 91 and 100% for the Au and Ag NP number concentrations. Whereas analysis mode -III showed a recovery of 70-88% for the same materials. Because of the polydispersity, the small size and polyhedral shape of the CeO2 NPs it was not possible to make NP number concentration comparisons for this material.