Results of a short study of interactions of cetaceans and longline fisheries in Atlantic waters: environmental correlates of catches and depredation events

In the Atlantic, economic losses have been reported from shark, swordfish and tuna longline fisheries due to depredation by cetaceans. We examined interactions of odontocete cetaceans with commercial longliners operating in waters off Brazil and the Azores archipelago during 2006–2007, analysing relationships between catches, depredation on hooked fish, cetacean sightings, acoustic records of cetacean presence and environmental variables. Data were provided by skippers of six vessels and by on-board observers for two vessels. The percentage of longline sets depredated by cetaceans was low (ranging from 1% to 9% of total sets per ship) but the proportion of fish damaged was high (up to 100%) when depredation occurred. Catches were related to the phase of the moon, cloud cover, sea surface temperature and water depth whereas cetacean sightings were primarily related to catches. In particular there was a positive association between Delphinus delphis sightings and catches of swordfish, and between Stenella frontalis sightings and mako catches. Acoustic detection was low when depredation by false killer whales occurred although high rates of clicks were detected when delphinids were sighted and false killer whales were by-caught. This may indicate that false killer whales are not echolocating when feeding on fish hooked on a longline. Guest editor: V. D. Valavanis Essential Fish Habitat Mapping in the Mediterranean     

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions.     

Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity acclimation

Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlantic coast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order to deal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural, biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmental salinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarine adaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity on N. granulata responses at the ecological, organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structural adaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, this review compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.     

Effect of microcystin on ion regulation and antioxidant system in gills of the estuarine crab Chasmagnathus granulatus (Decapoda,Grapsidae)

The objective of this work was to evaluate mechanisms of microcystin toxicity on crustacean species. Adult male crabs of Chasmagnathus granulatus (13.97+/-0.35 g) acclimated to low salinity (2 per thousand ) were injected with saline (control) or Microcystis aeruginosa aqueous extract (39.2 microg/l) at 24 h intervals for 48 h. After the exposure period, the anterior and posterior gills were dissected, measuring Na(+),K(+)-ATPase and glutathione-S-transferase (GST) activity. Total oxyradical scavenging capacity (TOSC) and lipid peroxides (LPO) content were also determined. Na(+),K(+)-ATPase activity in anterior gills was significantly lower in crabs injected with toxin than in control crabs, while no significant difference in the enzyme activity was detected in posterior gills. Both sodium and chloride concentration in the hemolymph were not affected by toxin exposure. Significant changes in GST activity were detected in posterior gills, with higher values being observed in the toxin-injected crabs. Crabs exposed to microcystin also showed a significant increase in the TOSC value against peroxyl radicals, for both anterior and posterior gills. Lipid peroxides level did not change in both gill types after exposure to the toxin. The increased levels of TOSC suggest the occurrence of a crab response against oxidative stress induced by toxin injection, which prevents lipid peroxidation.     

Carbon catabolite regulation of secondary metabolite formation,an old but not well-established regulatory system

Secondary microbial metabolites have various functions for the producer microorganisms, which allow them to interact and survive in adverse environments. In addition to these functions, other biological activities may have clinical relevance, as diverse as antimicrobial, anticancer and hypocholesterolaemic effects. These metabolites are usually formed during the idiophase of growth and have a wide diversity in their chemical structures. Their synthesis is under the impact of the type and concentration of the culture media nutrients. Some of the molecular mechanisms that affect the synthesis of secondary metabolites in bacteria (Gram-positive and negative) and fungi are partially known. Moreover, all microorganisms have their peculiarities in the control mechanisms of carbon sources, even those belonging to the same genus. This regulatory knowledge is necessary to establish culture conditions and manipulation methods for genetic improvement and product fermentation. As the carbon source is one of the essential nutritional factors for antibiotic production, its study has been imperative both at the industrial and research levels. This review aims to draw the utmost recent advances performed to clarify the molecular mechanisms of the negative effect exerted by the carbon source on the secondary metabolite formation, emphasizing those found in Streptomyces, one of the genera most profitable antibiotic producers.     

Genetic variation in tumor necrosis factor and lymphotoxin-alpha (TNF–LTA) and breast cancer risk

Tumor necrosis factor (TNF) is critical to regulation of inflammation. Genetic variation in the promoter region of TNF has been associated with expression differences, and a range of auto-immune, infectious, and oncologic diseases. We analyzed eight common single nucleotide polymorphisms (SNPs) (rs746868, rs909253, rs1799964, rs1800630, rs1800750, rs1800629, rs361525, and rs1800610) to capture most of the genetic variation in TNF in addition to SNPs in lymphotoxin-alpha (LTA), a pro-inflammatory cytokine in linkage disequilibrium with the TNF promoter region. SNPs were genotyped in a USA population-based case-control study (3,318 cases, 2,841 controls). Promising results were followed-up in an independent population-based case-control study in Poland (2,228 cases, 2,378 controls). In both studies, women carrying the variant allele of rs361525 were at elevated breast cancer risk compared to the GG genotype (per allele OR = 1.18, 95% CI 1.04–1.35; P for trend = 0.008). Other SNPs were not significantly associated with breast cancer risk. Haplotype analyses did not reveal any additional associations between TNF and breast cancer risk. Data from 5,269 cases and 4,982 controls suggested that the rs361525 A allele, located in the TNF promoter region, was associated with a modest increase in breast cancer risk. Additional studies are required to replicate these findings and to determine whether rs361525 is a causative SNP or is a marker of a causative SNP.     

Micronuclei induced by airborne particulate matter from Mexico City

Particulate air pollution is an important environmental health risk. In the present study, we have investigated the ability of chemically characterized water and organic-soluble extracts of PM(10) from two different regions of Mexico City to induce micronuclei in a human epithelial cell line. We also evaluated the association between the chemical characteristics of the PM and its genotoxicity. The airborne particulate samples were collected from an industrial and a residential region; a Hi-Vol air sampler was used to collect PM(10) on glass fiber filters. PM mass was determined by gravimetric analysis of the filters. One section of each PM(10) filter was agitated either with deionized water to extract water-soluble compounds or with dichloromethane to prepare organic-soluble compounds. The chemical composition of the extracts was determined by ion and gas chromatography and atomic adsorption spectroscopy. A549-human alveolar epithelial cells were exposed to different concentrations of PM(10) extracts and the cytokinesis blocked micronucleus assay was performed to measure DNA damage. Even though the industrial region had a higher PM concentration, higher amounts of metals and PAHs were found in the residential area. Both industrial and residential extracts induced a significant concentration-related increase in the micronuclei frequency. The PM(10) water-soluble industrial extract induced significantly more micronuclei than the one of the residential region; inversely, the organic residential extract induced more micronuclei than the one from the industrial region. The association between the induction of micronuclei and the chemical components obtained by the comparative analysis of standardized regression coefficients showed that cadmium and PAHs were significantly associated with micronuclei induction. Data indicate that water-soluble metals and the organic-soluble fraction of PM(10) are both important in the production of micronuclei. Effects observed, point to the risk of PM exposure and shows the need of integrative studies.     

Nuclear translocation of β-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype

Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.