The rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ...ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).
The metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, γ-H2AX and RAD51 foci formation).
We show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles.
We conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.
Circadian clocks in the brain and peripheral tissues temporally coordinate local physiology to align with the 24 hours rhythmic environment through light/darkness, rest/activity and feeding/fasting ...cycles. Circadian disruptions (during ageing, shift work and jet-lag) have been proposed as a risk factor for degeneration and disease of tissues, including the musculoskeletal system. The intervertebral disc (IVD) in the spine separates the bony vertebrae and permits movement of the spinal column. IVD degeneration is highly prevalent among the ageing population and is a leading cause of lower back pain. The IVD is known to experience diurnal changes in loading patterns driven by the circadian rhythm in rest/activity cycles. In recent years, emerging evidence indicates the existence of molecular circadian clocks within the IVD, disruption to which accelerates tissue ageing and predispose animals to IVD degeneration. The cell-intrinsic circadian clocks in the IVD control key aspects of physiology and pathophysiology by rhythmically regulating the expression of ~3.5% of the IVD transcriptome, allowing cells to cope with the drastic biomechanical and chemical changes that occur throughout the day. Indeed, epidemiological studies on long-term shift workers have shown an increased incidence of lower back pain. In this review, we summarise recent findings of circadian rhythms in health and disease, with the IVD as an exemplar tissue system. We focus on rhythmic IVD functions and discuss implications of utilising biological timing mechanisms to improve tissue health and mitigate degeneration. These findings may have broader implications in chronic rheumatic conditions, given the recent findings of musculoskeletal circadian clocks.
Plastic debris has been recognized as a growing threat to marine biota due to its widespread distribution and possible interactions with marine species. Concerns over the effects of plastic polymers ...in marine ecosystems is reflected in the high number of toxicological studies, regarding microplastics (<5 mm) and marine fauna. Although several studies reported that organisms ingest and subsequently eliminate microplastics (MP), the potential effects at organ and tissue level remain unclear, especially considering exposure to different microplastic sizes and concentrations. The present study aimed at investigating potential pathophysiological effects of the ingestion of MP by marine filter-feeders. For the purpose, Mediterranean mussel (Mytilus galloprovincialis) was exposed to spherical polystyrene MP (2 and 10 μm Ø) over short- and medium-term exposure periods, under single and combined concentrations that represent high, yet realistic doses (10 and 1000 MP mL−1). Overall, results suggest rapid MP’ clearance from water column by filtering, regardless of MP size. Ingestion occurred, identified by MP in the lumen of the gut (mostly in midgut region), followed by excretion through faeces. However, no MP were found in gills or digestive gland diverticula. Biochemical indicators for oxidative stress were generally irresponsive regardless of organ and time of exposure. Small foci of haemocytic infiltration in gastric epithelia were found, albeit not clearly related to MP ingestion. Globally, no evident histopathological damage was recorded in whole-body sections of exposed animals. The present findings highlight the adaptative ability of filter-feeding bivalves to cope with filtration of suspended MP, resulting in rapid elimination and reduced internal damage following ingestion of spherical MP. Nevertheless, the fact that the animals are able to translocate MP to the gut reveals that filter feeding organisms may indeed became a target of concern for fragmented materials with smaller, mixed sizes and sharper edges.
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•Mussels are able to ingest and excrete polystyrene microplastics.•MP are rapidly processed through the mussels' digestive tract.•No significant pathological effects occurred even during longer-term exposure.•No clear evidence of oxidative stress caused by ingested polystyrene MP.
Effects of microplastic ingestion by filter-feeding bivalves.
Summary
Cyanobacteria were the first organisms ever to perform oxygenic photosynthesis and still significantly contribute to primary production on a global scale. To assure the proper functioning of ...their primary metabolism and cell homeostasis, cyanobacteria must rely on efficient transport systems to cross their multilayered cell envelope. However, cyanobacterial secretion mechanisms remain largely unknown. Here, we report on the identification of 11 putative inner membrane translocase components of TolC‐mediated secretion in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Gene‐inactivation of each of the candidate genes followed by a comprehensive phenotypic characterization allowed to link specific protein components to the processes of protein export (as part of the type I secretion system) and drug efflux (part of the resistance‐division‐nodulation efflux pumps). In addition, mutants in genes sll0141, sll0180 and slr0369 exhibited alterations in pilin glycosylation, but pili structures could still be observed by transmission electron microscopy. By studying the release of outer membrane vesicles (OMVs), an alternative secretion route, on mutants with impaired secretory functions we suggest that the hyper‐vesiculating phenotype of the TolC‐deficient mutant is related to cell envelope stress management. Altogether, these findings highlight how both classical (TolC‐mediated) and nonclassical (OMVs‐mediated) secretion systems are crucial for cyanobacterial cell homeostasis.
Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light and feeding). The presence of tissue ...niche-dependent physiological time cues has been proposed, allowing tissues the ability of circadian phase adjustment based on local signals. However, to date, such stimuli have remained elusive. Here we show that daily patterns of mechanical loading and associated osmotic challenge within physiological ranges reset circadian clock phase and amplitude in cartilage and intervertebral disc tissues in vivo and in tissue explant cultures. Hyperosmolarity (but not hypo-osmolarity) resets clocks in young and ageing skeletal tissues and induce genome-wide expression of rhythmic genes in cells. Mechanistically, RNAseq and biochemical analysis revealed the PLD2-mTORC2-AKT-GSK3β axis as a convergent pathway for both in vivo loading and hyperosmolarity-induced clock changes. These results reveal diurnal patterns of mechanical loading and consequent daily oscillations in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.
The aim of this study is the further characterisation of PM₂.₅ emissions from the residential wood combustion of common woods grown in Portugal. This new research extends to eight the number of ...biomass fuels studied and tries to understand the differences that the burning appliance (fireplace versus woodstove) and the combustion temperature (cold and hot start) have on emissions. Pinus pinaster (Maritime pine), Eucalyptus globulus (eucalypt), Quercus suber (cork oak), Acacia longifolia (Golden wattle), Quercus faginea (Portuguese oak), Olea europea (Olive), Quercus ilex rotundifolia (Holm oak) and briquettes produced from forest biomass waste were used in the combustion tests. Determinations included fine particle emission factors, carbonaceous content (OC and EC) by a thermal–optical transmission technique and detailed identification and quantification of organic compounds by gas chromatography–mass spectrometry. Fine particle emission factors from the woodstove were lower than those from the fireplace. For both combustion appliances, the OC/EC ratio was higher in “cold start” tests (1.56 ± 0.95 for woodstove and 2.03 ± 1.34 for fireplace). These “cold start” OC/EC values were, respectively, for the woodstove and the fireplace, 51% and 69% higher than those obtained in “hot start” experiments. The chromatographically resolved organics included n-alkanes, n-alkenes, PAHs, n-alkanals, ketones, n-alkanols, terpenoids, triterpenoids, phenolic compounds, phytosterols, alcohols, n-alkanoic acids, n-di-acids, unsaturated acids and alkyl esters of acids. The smoke emission rate and composition varied widely depending on fuel type, burning appliance and combustion temperature.
On May 2009, both the gas and particulate fractions of smoke from a wildfire in Sever do Vouga, central Portugal, were sampled. Total hydrocarbons and carbon oxides (CO2 and CO) were measured using ...automatic analysers with flame ionisation and non-dispersive infrared detectors, respectively. Fine (PM2.5) and coarse (PM2.5–10) particles from the smoke plume were analysed by a thermal–optical transmission technique to determine the elemental and organic carbon (EC and OC) content. Subsequently, the particle samples were solvent extracted and fractionated by vacuum flash chromatography into different classes of organic compounds. The detailed organic speciation was performed by gas chromatography–mass spectrometry. The CO, CO2 and total hydrocarbon emission factors (gkg−1 dry fuel) were 170±83, 1485±147, and 9.8±0.90, respectively. It was observed that the particulate matter and OC emissions are significantly enhanced under smouldering fire conditions. The aerosol emissions were dominated by fine particles whose mass was mainly composed of organic constituents, such as degradation products from biopolymers (e.g. levoglucosan from cellulose, methoxyphenols from lignin). The compound classes also included homologous series (n-alkanes, n-alkenes, n-alkanoic acids and n-alkanols), monosaccharide derivatives from cellulose, steroid and terpenoid biomarkers, and polycyclic aromatic hydrocarbons (PAHs). The most abundant PAH was retene. Even carbon number homologs of monoglycerides were identified for the first time as biomarkers in biomass burning aerosols.
► Wildfires release into the atmosphere tons of gaseous and particulate pollutants. ► Wildfire emission factors for the Mediterranean region have not yet been obtained. ► Emission factors for trace gases and smoke aerosols were estimated. ► Several biomarkers in biomass burning aerosols were quantified.
Either through differentiated glands or specialised individual cells, the coating epithelia of soft-bodied marine invertebrates are responsible for the secretion of a broad span of peptidic ...substances, from protective mucins to biocides. These secretions are characterised by the presence of cysteine-rich proteins and peptides, rendering a distinct histochemical signature of secretory epithelia. Through a histochemical procedure for fluorescence microscopy in paraffin sections, we performed a comparative assessment of the distribution of thiol-rich compounds in multiple epithelia of different species of intertidal Polychaeta, which revealed distinctive patterns of distribution that closely relate to ecology, morphoanatomy and physiology. The presence of free thiols was notorious in mucocytes and enzyme-plus toxin-secreting cells. Consequently, strong signals were recorded in the mucocytes of the parapodia of
Nereis splendida
, the epidermis and pharynx epithelium of
Mysta picta
and the venom glands of
Glycera alba
. The findings show an investment in mucus secretion in foragers such as
Nereis
and
Mysta
, especially the latter, which is not a native burrower, as a protective response and as lubricant for locomotion. Additionally, nereidids are believed to secret integumentary toxins for defence. On the other hand,
Glycera
is an ambush predatorial burrower whose behaviour entirely revolves around the delivery of venom making use of its four jaws. The results showed that the detection of thiol-rich compounds in histological sections can be a tool to identify potential toxin secretion and delivery structures, with important consequences for the bioprospecting of novel bioreactives from marine invertebrates for the purpose of drug discovery.
Studies in shift workers and model organisms link circadian disruption to breast cancer. However, molecular circadian rhythms in noncancerous and cancerous human breast tissues and their clinical ...relevance are largely unknown. We reconstructed rhythms informatically, integrating locally collected, time-stamped biopsies with public datasets. For noncancerous breast tissue, inflammatory, epithelial-mesenchymal transition (EMT), and estrogen responsiveness pathways show circadian modulation. Among tumors, clock correlation analysis demonstrates subtype-specific changes in circadian organization. Luminal A organoids and informatic ordering of luminal A samples exhibit continued, albeit dampened and reprogrammed rhythms. However, CYCLOPS magnitude, a measure of global rhythm strength, varied widely among luminal A samples. Cycling of EMT pathway genes was markedly increased in high-magnitude luminal A tumors. Surprisingly, patients with high-magnitude tumors had reduced 5-y survival. Correspondingly, 3D luminal A cultures show reduced invasion following molecular clock disruption. This study links subtype-specific circadian disruption in breast cancer to EMT, metastatic potential, and prognosis.
In the early fall of 2010 a survey questionnaire was conducted to assess residential wood combustion (RWC) practices in the 18 districts of mainland Portugal. This paper describes and analyses the ...results from a bottom-up approach, based on this telephone survey, which enabled the characterisation of RWC practices in each district. Additionally, emission factors and source profiles obtained from different RWC tests, allowed the estimation of CO, CO2, PM2.5 and particulate organic compound emissions on a country-scale. The type and number of RWC appliances, each with their specific emissions factor and the amount and type of fuel used vary significantly on a district scale. The energy consumption in RWC appliances was estimated to be 35 342 TJ y−1. The estimated emissions of PM2.5 from RWC in Portugal (10.96 kt y−1) represent 30% of the estimate for different source activities reported in a recent inventory. The national emissions of OC and EC were estimated to be 5.32 and 0.53 kt y−1, respectively. It was also estimated that the total CO2 emissions from RWC in Portugal are 1.7 Mt y−1. The levoglucosan emissions contribute with between 7.8% and 16.1% of PM2.5 emissions from RWC.
► RWC is one of the largest sources of fine particle emissions in Portugal. ► An assessment of RWC practices was made through a national scale survey. ► Total of wood used in Portugal in 2010 was 1.95 Mt for heating and cooking purposes. ► The estimated emissions of PM2.5 from RWC in Portugal were 10.96 kt y−1. ► The global levoglucosan emissions for Portugal were estimated to be 1.2 kt y−1.
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