Physiological response to persistent organic pollutants in fish from mountain lakes: analysis of CYP1A gene expression in natural populations of Salmo trutta

Cytochrome p450 1A (CYP1A) gene expression in fish liver increases upon exposure to a variety of chemical compounds, including organochlorine compounds (OCs) and polycyclic aromatic hydrocarbons (PAHs). To use this physiological response as a marker of environmental impact, we developed and validated a set of primers to quantify CYP1A expression by qRT-PCR in the brown trout, Salmo trutta. These primers were used to explore the natural variability of CYP1A expression in 8 isolated populations (65 samples) from European remote lakes, in a geographical distribution encompassing the Tyrolean Alps, Pyrenees, Rila, Tatras, and Norwegian and Scottish mountains. CYP1A expression values varied more than 2 orders of magnitude among samples, with strong variations within each population. CYP1A expression values were significantly elevated in Tatras and Pyrenees fish populations, whereas the lowest median values were found in populations from the Tyrolean Alps and Rila. These values correlated with the content of different environmentally relevant pollutants in the sediments of the lakes harboring each fish population, particularly with HCB and 4,4'-DDE contents. To our knowledge, this works represents a first report of a physiological response linked to persistent organic pollutants in fish from mountain lakes.     

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

ICA1L Is Associated with Small Vessel Disease: A Proteome-Wide Association Study in Small Vessel Stroke and Intracerebral Haemorrhage

Small vessel strokes (SVS) and intracerebral haemorrhages (ICH) are acute outcomes of cerebral small vessel disease (SVD). Genetic studies combining both phenotypes have identified three loci associated with both traits. However, the genetic cis-regulation at the protein level associated with SVD has not been studied before. We performed a proteome-wide association study (PWAS) using FUSION to integrate a genome-wide association study (GWAS) and brain proteomic data to discover the common mechanisms regulating both SVS and ICH. Dorsolateral prefrontal cortex (dPFC) brain proteomes from the ROS/MAP study (N = 376 subjects and 1443 proteins) and the summary statistics for the SVS GWAS from the MEGASTROKE study (N = 237,511) and multi-trait analysis of GWAS (MTAG)-ICH–SVS from Chung et al. (N = 240,269) were selected. We performed PWAS and then a co-localization analysis with COLOC. The significant and nominal results were validated using a replication dPFC proteome (N = 152). The replicated results (q-value < 0.05) were further investigated for the causality relationship using summary data-based Mendelian randomization (SMR). One protein (ICA1L) was significantly associated with SVS (z-score = −4.42 and p-value = 9.6 × 10⁻⁶) and non-lobar ICH (z-score = −4.8 and p-value = 1.58 × 10⁻⁶) in the discovery PWAS, with a high co-localization posterior probability of 4. In the validation PWAS, ICA1L remained significantly associated with both traits. The SMR results for ICA1L indicated a causal association of protein expression levels in the brain with SVS (p-value = 3.66 × 10⁻⁵) and non-lobar ICH (p-value = 1.81 × 10⁻⁵). Our results show that the association of ICA1L with SVS and non-lobar ICH is conditioned by the cis-regulation of its protein levels in the brain.     

Discovery of novel non-cyclam polynitrogenated CXCR4 coreceptor inhibitors

HIV cell fusion and entry have been validated as targets for therapeutic intervention against infection. Bicyclams were the first low-molecular-weight compounds to show specific interaction with CXCR4. The most potent bicyclam was AMD3100, in which the two cyclam moieties are tethered by a 1,4-phenylenebis(methylene) bridge. It was withdrawn from clinical trials owing to its lack of oral bioavailability and cardiotoxicity. We have designed a combinatorial library of non-cyclam polynitrogenated compounds by preserving the main features of AMD3100. At least two nitrogen atoms on each side of the p-phenylene moiety, one in the benzylic position and the other(s) in the heterocyclic system were maintained, and the distances between them were similar to the nitrogen atom distances in cyclam. A selection of diverse compounds from this library were prepared, and their in vitro activity was tested in cell cultures against HIV strains. This led to the identification of novel potent CXCR4 coreceptor inhibitors without cytotoxicity at the tested concentrations.     

Tau Exon 10 Inclusion by PrPC through Downregulating GSK3β Activity

Tau protein is largely responsible for tauopathies, including Alzheimer’s disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3β, whose activity is inhibited by the cellular prion protein (PrP^C), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrP^C and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrP^C and the implication of GSK3β in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrP^C ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrP^C expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrP^C levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3β activity downstream from PrP^C in this phenomenon. Our results indicate that PrP^C plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3β.     

Adenosine Signaling in Mast Cells and Allergic Diseases

Adenosine is a nucleoside involved in the pathogenesis of allergic diseases. Its effects are mediated through its binding to G protein-coupled receptors: A1, A2a, A2b and A3. The receptors differ in the type of G protein they recruit, in the effect on adenylyl cyclase (AC) activity and the downstream signaling pathway triggered. Adenosine can produce both an enhancement and an inhibition of mast cell degranulation, indicating that adenosine effects on these receptors is controversial and remains to be clarified. Depending on the study model, A1, A2b, and A3 receptors have shown anti- or pro-inflammatory activity. However, most studies reported an anti-inflammatory activity of A2a receptor. The precise knowledge of the adenosine mechanism of action may allow to develop more efficient therapies for allergic diseases by using selective agonist and antagonist against specific receptor subtypes.     

Analysis of the HLA class I associated peptide repertoire in a hepatocellular carcinoma cell line reveals tumor-specific peptides as putative targets for immunotherapy

HLA class I molecules present peptides on the cell surface to CD8(+) T cells. The repertoire of peptides that associate to class I molecules represents the cellular proteome. Therefore, cells expressing different proteomes could generate different class I-associated peptide repertoires. A large number of peptides have been sequenced from HLA class I alleles, mostly from lymphoid cells. On the other hand, T cell immunotherapy is a goal in the fight against cancer, but the identification of T cell epitopes is a laborious task. Proteomic techniques allow the definition of putative T cell epitopes by the identification of HLA natural ligands in tumor cells. In this study, we have compared the HLA class I-associated peptide repertoire from the hepatocellular carcinoma (HCC) cell line SK-Hep-1 with that previously described from lymphoid cells. The analysis of the peptide pool confirmed that, as expected, the peptides from SK-Hep-1 derive from proteins localized in the same compartments as in lymphoid cells. Within this pool, we have identified 12 HLA class I peptides derived from HCC-related proteins. This confirms that tumor cell lines could be a good source of tumor associated antigens to be used, together with MS, to define putative epitopes for cytotoxic T cells from cancer patients.     

Segmentation of multiple sclerosis lesions in brain MRI: A review of automated approaches

Automatic segmentation of multiple sclerosis (MS) lesions in brain MRI has been widely investigated in recent years with the goal of helping MS diagnosis and patient follow-up. However, the performance of most of the algorithms still falls far below expert expectations. In this paper, we review the main approaches to automated MS lesion segmentation. The main features of the segmentation algorithms are analysed and the most recent important techniques are classified into different strategies according to their main principle, pointing out their strengths and weaknesses and suggesting new research directions. A qualitative and quantitative comparison of the results of the approaches analysed is also presented. Finally, possible future approaches to MS lesion segmentation are discussed.     

Input–output analysis of alternative policies implemented on the energy activities: An application for Catalonia

The aim of this paper is to analyze the economic impact of alternative policies implemented on the energy activities of the Catalan production system. Specifically, we analyze the effects of a tax on intermediate energy uses, a reduction in intermediate energy demand, and a tax on intermediate uses combined with a reduction in intermediate energy demand. The methodology involves two versions of the input–output price model: a competitive price formulation and a mark-up price formulation. The input–output price framework will make it possible to evaluate how the alternative measures modify production prices, consumption prices, private real income, and intermediate energy uses. The empirical application is for the Catalan economy and uses economic data for the year 2001. The combination of a tax on energy uses and an improvement in the energy efficiency of the production system is a measure that accomplishes both economic and environmental goals, since it has no effects on prices, it has a positive effect on private real income and, finally, energy consumption is considerably reduced.     

Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members’ follow-up.