Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation.

Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.

A Lipophilic 4-Phenylbutyric Acid Derivative That Prevents Aggregation and Retention of Misfolded Proteins.

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its unfavorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2-isopropyl-4-phenylbutanoic acid (5) has been found to be 2-10-fold more effective than 4-PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).

First evidence of microplastics ingestion in benthic amphipods from Svalbard.

The present paper provides the first record of ingestion of microplastics in natural context by Gammarus setosus from Svalbard Archipelago. The plastic particles were identified both by Nile Red staining and Micro FT-IR spectroscopy. The species studied ingests microplastic particles in natural conditions if present in its habitat, probably mistaking them as food. The microplastic particles ingested may be available for uptake to predators that consume this Arctic amphipod, producing consequences to the food web.

Evaluation of Genotoxic Potential of Waters from Two Italian Rivers in Gammarus elvirae (Amphipoda).

The aim of the present work is to evaluate the genotoxic impact of contaminants along the whole course of Ninfa-Sisto and Amaseno (Latium, Italy) rivers. The authors performed the alkaline Comet assay to assess DNA damage in the freshwater amphipod Gammarus elvirae, exposed ex situ for 24 hours and 7 days to water collected at different sites. The assay, applied on haemocytes, provides a sensitive tool to reveal effects even at low concentrations of pollutants. The results indicate significant increase of DNA damage along the course of the two rivers, compared to the unpolluted upstream sites, even if the analytes do not exceed the permissible limits. Moreover, the results show that there is not a linear correlation between the concentration of analytes and DNA damage. Based on this study's results, it would be desirable to use Comet assay, on proposed test species, as an early warning method to detect genotoxic potential of waters.

Molecular and cellular responses to short exposure to bisphenols A, F, and S and eluates of microplastics in C. elegans.

Bisphenol F (BPF) and bisphenol S (BPS) have been developed as an alternative to bisphenol A (BPA), a well-known endocrine disruptor, leading to their detection in the aquatic environment. In this work, we used the animal model Caenorhabditis elegans to improve our understanding of their potential effects on the biota and the environment. Our findings demonstrated that, after 24 h exposure, all the bisphenols examined increased the number of apoptotic corpses and the expression of the detoxifying enzymes SOD-3 and GST-4, without affecting the ROS levels, while BPA and BPS significantly enhanced DNA fragmentation. Furthermore, similarly to BPA, BPF and BPS did not alter the lifespan through the activation of SEK-1 and SKN-1 pathways. Thus, this study raises the attention of the risks associated with exposure to BPA alternatives. We also examined the effects of microplastic (MP) eluates on C. elegans. Aqueous extracts of weathered microplastic samples, both at high and low degradation state and pellets, have been evaluated for their effects on lifespan, DNA fragmentation, germline apoptosis, and oxidative stress response. Overall, our findings showed that eluates of low degraded plastics exert a greater toxic effect on the nematode C. elegans compared with the aqueous sample of high degraded plastic fragments and pellets.

Plastic abundance and seasonal variation on the shorelines of three volcanic lakes in Central Italy: can amphipods help detect contamination?

Despite the exponential increase of studies on plastic debris in recent years, there are still few works focusing on the problem as it relates to inland waters: little is known about the accumulation and dispersion dynamics on lake shores, and there are no standardized sampling methods for monitoring purposes. The accumulation of plastic litter in natural habitats also threatens the resident organisms. In this paper, we investigated the abundance and accumulation of plastic particles, ranging in size from 1 to 50 mm, from the beach sediment of three volcanic lakes in Central Italy: Albano, Bracciano, and Vico. The collection was designed to define the most important variables that one must consider in order to obtain a representative sample of plastic litter in a lake environment. In view of the high heterogeneity of sampling protocols used, comparison among the obtained results is limited and sometimes impossible. By using one of the proposed sampling methodologies, and critically analyzing the results, we aimed to highlight a possible monitoring criterion and to identify specific elements that can be meaningful and representative. The samples were collected in May and September 2017. For each lake, we sampled plastic items and sediments from two beaches. Albano contained the largest amount of plastic (in weight), while Bracciano had the largest number of particles. Our observations lead us to infer that the number of particles is the parameter most sensitive to environmental variations, as well as the more suitable for monitoring with greater definition the differences between sites. Moreover, sampling should be taken in different seasons, following a sampling pattern that includes at least two beaches placed in strategic positions with respect to wind and waves.In order to identify new indicators to evaluate the entry points of plastic into the food web, we collected, from the same sites analyzed, some specimens of the Talitrid Amphipod Cryptorchestia garbinii, a detritivorous species having a critical role in debris turnover of these environments. To investigate the microplastic (MP) ingestion in natural conditions, we analyzed their digestive tracts with both Nile red staining method and micro-FTIR spectroscopy. The analyses confirmed that C. garbinii was able to ingest plastics in natural conditions. Therefore, it can signify one of the entry points for microplastics (MPs) in the trophic chain. This observation constitutes the first evidence of MP ingestion in this species.

Effect of short-time exposures to nickel and lead on brain monoamine oxidase from Danio rerio and Poecilia reticulata.

The aim of this work was to verify, in two small size freshwater teleosts Danio rerio and Poecilia reticulata, the effects of short-time exposures (24 and 72 h) to a sublethal dose (500 microg/L) of nickel and lead, on brain monoamine oxidase (MAO), an important neural enzyme. The 24-h treatment using both metals caused a strong reduction of MAO activity in D. rerio brain, whereas causing a slight MAO activity stimulation in P. reticulata brain. The same treatment in both species did not affect the brain MAO mRNA production as showed by RT-PCR. Extending the duration of treatment as far as 72 h, partly (D. rerio) or completely (P. reticulata) reversed the metal effects on brain MAO activity suggesting that mechanisms to neutralize the metals had been activated.

In-vivo assesment of the genotoxic and oxidative stress effects of particulate matter on Echinogammarus veneris.

Seven types of atmospheric dusts (road dust, soil dust, brake dust, desert dust, pellet ash and coke and certified material NIST1648a - urban dust) have been tested for their genotoxicity on specimens of Echinogammarus veneris, a small aquatic amphipod. Experiments were carried out in vivo, by exposing the animals for 24 h to water containing 25 mg/L of dust. Each dust has been chemically analyzed for ions, elemental carbon, organic carbon and for the soluble and insoluble fractions of elements. Non-specific damages to DNA have been evaluated by the comet test, while oxidative damages have been estimated by coupling the comet test with formamido pyrimidine DNA glycosylase reaction. The animal tissues have been acid digested and analyzed for their elemental content to evaluate the bioaccumulation. All the considered dusts have caused a significant non-specific DNA damage, while the oxidative stress was shown only by dust types containing high concentration of elements. Furthermore, the oxidative damage has shown a positive correlation with the total bio-accumulated elemental concentration. For all the dust samples, the correlation with bio-accumulation in the tissues was more satisfactory for the insoluble fraction than for the soluble fraction of elements. Elements contained in solid particles seem then to be the main responsible bioaccumulation and for the oxidative stress.

Molecular characterization of monoamine oxidase in zebrafish (Danio rerio).

Monoamine oxidase (MAO) is responsible for the degradation of a number of neurotransmitters and other biogenic amines. In terrestrial vertebrates, two forms of the enzyme, named MAO A and B, were found in which mammals are coded by two similar but distinct genes. In teleosts, the biochemical data obtained so far indicate that enzyme activity is due to a single form, whose sequence, obtained for trout, displays 70% identity with mammal MAO A and B. In this paper, we carried out an investigation of zebrafish MAO (Z-MAO) to shed further light on the nature of the MAO form present in aquatic vertebrates. Sequencing studies have revealed an open reading frame 522-amino-acids long with MW 58.7 kDa, displaying 84% identity with trout MAO and about 70% identity with mammal MAO A and MAO B. Analysis of the sequence and of the predicted secondary structure shows that also in Z-MAO principal domains characterizing the MAOs are present. The domain linking the FAD is very well conserved, while the transmembrane domain sequence linking the enzyme to the external mitochondrial membrane does not appear to be conserved even with respect to trout MAO. Comparison with the amino acids which, according to the human MAO B and rat MAO A models, line the substrate-binding site shows that in Z-MAO, several residues (V172, N173, F200, L327) differ from MAO B but are similar or identical to the corresponding ones present in rat MAO A, as well as in trout MAO. A three-dimensional model is reported of the substrate-binding site of Z-MAO obtained by comparative modeling. Our observations support the hypothesis that the MAO form present in aquatic vertebrates is a MAO A-like form. Experiments performed to test the effect of selective MAO A (clorgyline) and MAO B (deprenyl) inhibitors on the enzyme's activity in liver and brain confirm the presence of a single form of MAO in zebrafish.

An integrated study on Gammarus elvirae (Crustacea, Amphipoda): perspectives for toxicology of arsenic-contaminated freshwater.

The Italian region Latium is characterized by extensive quaternary volcanic systems that contribute greatly to arsenic (As) contamination of freshwater, including drinking water supplies. However, knowledge of the possible toxic effects in these aquatic environments is, despite being highly relevant to public health, still limited. In this paper, we approach this issue using Gammarus elvirae, an amphipod species that inhabits rivers and streams in central Italy, including Latium. We explored the possibility of using G. elvirae in the toxicology of freshwater by addressing the most relevant issues. First, we tested the usefulness of hemocytes from G. elvirae in determining non-specific DNA damage by means of the Comet assay after exposure (24 h and 7 days) to different river water samples in Latium; second, we provided an interpretative overview of the usefulness of hepatopancreatic epithelial cells of G. elvirae as a means of assessing toxicity after long-term exposure to As and other pollutants; third, the LC (50-240 h) value for G. elvirae was estimated for arsenate, which is usually the dominant arsenic species in surface waters. Our study sheds light on G. elvirae at different levels, providing a background for future toxicological research of freshwater.

Genotoxic risk and oxidative DNA damage in workers exposed to antimony trioxide.

The growing use of antimony (Sb) compounds in industry and the consequent increase in the number of exposed workers make it important to carry out a health risk assessment. The main goal of this study was to assess the genotoxicity of Sb(2)O(3) in occupationally exposed workers. Genotoxicity was evaluated by the sister chromatid exchange (SCE) and micronucleus tests, and the enzyme (Fpg)-modified comet assay. In addition, antimony exposure levels were established by environmental monitoring with personal air samplers. We studied 23 male workers assigned to different fire retardant treatment tasks in the car upholstery industry and a control group of 23 healthy nonexposed males. The exposed workers were divided into two groups on the basis of their tasks and the work cycle: Group A comprised finishing and intermediate inspection operators who directly handled a mixture containing Sb(2)O(3); Group B were jet operators, not directly exposed to the compound. Environmental monitoring detected low Sb exposure levels but significant differences between the two groups, with Group A having the higher exposure level. Cytogenetic analyses showed no difference between exposed workers and controls for micronuclei and SCE. The enzyme-modified comet assay showed a probable relation between moderate levels of oxidative DNA damage and exposure to antimony, with a significantly higher proportion of workers in Group A having oxidative DNA damage compared to controls. The results support the theory that oxidative DNA damage is involved in the genotoxicity of antimony and indicate the need for further research in this field.

Evaluation of DNA damage in flight personnel by Comet assay.

There have been some suggestions that air-crew are at a higher-than-normal risk of developing cancer, since they are exposed to potential genotoxic factors. These include cosmic radiations, airborne pollutants such as the combustion products of jet propulsion, ozone, and electromagnetic fields. We used the Comet assay to investigate DNA damage in flight personnel with the aim of assessing potential health hazards in this occupational category. We studied 40 civil air-crew members who had been flying long-haul routes for at least 5 years, and compared them with a homogeneous control group of 40 healthy male ground staff. The Comet assay, or single-cell gel electrophoresis (SCGE), detects DNA single- and double-strand breaks (DSBs) and alkali-labile lesions in individual cells, and is a powerful and sensitive technique for detecting genetic damage induced by different genotoxic agents. Taking into consideration occupational risk and possible confounding factors, this assay showed a small increase, that did not reach statistical significance, of DNA damage in long-haul crew members compared to controls, indicating a lack of evident genotoxic effects. An association, although again not statistically significant, was found between reduced DNA damage and use of protective drugs (antioxidants).

N-linked glycosylation selectively regulates the generic folding of HLA-Cw1.

To resolve primary (glycosylation-assisted) from secondary (glycosylation-independent) quality control steps in the biosynthesis of HLA (human leukocyte antigen) class I glycoproteins, the unique N-linked glycosylation site of the HLA-Cw1 heavy chain was deleted by site-directed mutagenesis. The non-glycosylated Cw1S88G mutant was characterized by flow cytometry, pulse-chase, co-immunoprecipitation, and in vitro assembly assays with synthetic peptide ligands upon transfection in 721.221 and 721.220 cells. The former provide a full set of primary as well as secondary chaperoning interactions, whereas the latter are unable to perform secondary quality control (e.g. proper class I assembly with peptide antigens) as a result of a functional defect of the HLA-dedicated chaperone tapasin. In both transfectants, Cw1S88G displayed a loss/weakening in its generic chaperoning interaction with calreticulin and/or ERp57 and became redistributed toward calnexin, known to bind the most unfolded class I conformers. Despite this, and quite unexpectedly, a weak interaction with the HLA-dedicated chaperone TAP was selectively retained in 721.221. In addition, the ordered, stepwise acquisition of thermal stability/peptide binding was disrupted, resulting in a heterogeneous ensemble of Cw1S88G conformers with unorthodox and unprecedented peptide assembly features. Because a lack of glycosylation and a lack of tapasin-assisted peptide loading have distinct, complementary, and additive effects, the former is separable from (and upstream of) the latter, e.g. primary quality control is suggested to supervise a crucial, generic folding step preliminary to the acquisition of peptide receptivity.

A single bottleneck in HLA-C assembly.

Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (beta(2)-microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-C-specific motif KYRV at residues 66-76 of the alpha1-domain alpha-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locus-specific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.

DNA damage and TNFalpha cytokine production in hairdressers with contact dermatitis.

The present work was undertaken to study in hairdressers exposed to several irritants and allergens (prevalently hair-dyeing) and affected by hand contact dermatitis the possible correlation between exposure and direct-oxidative DNA damage, production of tumour necrosis factor alpha (TNFalpha) and allergic inflammatory disease. We evaluated in 19 hairdressers with hand contact dermatitis, 14 allergic contact dermatitis (ACD) and 5 irritant contact dermatitis (ICD) and in a selected control group TNFalpha serum levels by ELISA and direct-oxidative DNA damage by Fpg (formamido-pyrimidine-glycosylase)-modified Comet test on blood. Hairdressers were divided on the basis of number of hair-dyeing carried out weekly into 2 groups: low-exposure (<60 hair-dyeing/week) and high-exposure hairdressers (>or=60 hair-dyeing/week) that reflect also the exposure to other allergens and irritants and 2 different tasks (hairdressers and apprentice hairdressers, respectively). Serum levels of TNFalpha in hairdressers with ACD were significantly higher than controls with a correlation to exposure level. Significant DNA damage in ICD hairdressers with higher exposure as compared to controls was found. These findings suggest that occupational exposure can induce in hairdressers, particularly ICD, DNA damage, increase the TNFa levels particularly in ACD and induce allergic sensitization, suggesting a relationship between direct-oxidative DNA damage, TNFalpha production and allergic inflammatory disease.

Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates.

Class I MHC H chains assemble with beta2-microglobulin (beta2m) and are loaded with peptide Ags through multiple folding steps. When free of beta2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, beta2m-free H chains with calnexin in beta2m-defective cells, and with calreticulin and TAP in beta2m-expressing cells. In beta2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the alpha1 domain alpha helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the alpha1 domain alpha helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.