Okadaic acid: chromosomal non-disjunction analysis in human lymphocytes and study of aneugenic pathway in CHO-K1 cells

Okadaic acid (OA) is the main marine toxin implicated in the diarrhetic shellfish poisoning (DSP) in humans after consumption of contaminated bivalve molluscs. We have previously shown that OA was an in vitro aneugenic compound that induced chromosome loss via micronuclei formation in CHO-K1 cells. The aims of this study were to investigate the chromosomal non-disjunction (ND) potential of OA in human lymphocytes and the pathways involved for aneuploidy in CHO-K1 cells. Firstly, we analysed the formation of micronuclei and the non-disjunction for chromosomes 1 and 17 in binucleated human lymphocytes cells with the cytokinesis-blocked micronucleus (CBMN) assay coupled to a fluorescent in situ hybridization (FISH) technique with centromere-specific DNA probes. We showed that OA statistically increased the frequency of micronucleated lymphocytes in the dose range from 20 to 35 nM. However, FISH analysis did not reveal any increase in the non-disjunction for both chromosomes whatever the concentration between 2.5 and 35 nM. However, a significant increase in ND for the chromosome 17 was found at 1 nM. Secondly, in CHO-K1 cells, we investigated the dose and time dependent effects of OA: (i) on cell cycle progression, (ii) on mitotic-phase arrest and (ii) on mitotic spindle and centrosome abnormalities. The results showed that OA induced a progressive accumulation of mitotic CHO-K1 cells in prometaphase, an induction of multipolar mitotic spindle with centrosome amplification and the formation of multinucleated cells. We concluded that OA did not induce chromosome non-disjunction but should more likely induced chromosome loss in human lymphocytes. Moreover, our results obtained in CHO-K1 suggest that OA induced aneuploidy by preventing the chromosome attachment to the mitotic spindle and by amplifying the centrosome. The mode of action of the toxin in relation to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A) and the mitosis process is discussed.     

Quantifying the effects of hydrogen on carbon assimilation in a seafloor microbial community associated with ultramafic rocks

Thermodynamic models predict that H₂ is energetically favorable for seafloor microbial life, but how H₂ affects anabolic processes in seafloor-associated communities is poorly understood. Here, we used quantitative ¹³C DNA stable isotope probing (qSIP) to quantify the effect of H₂ on carbon assimilation by microbial taxa synthesizing ¹³C-labeled DNA that are associated with partially serpentinized peridotite rocks from the equatorial Mid-Atlantic Ridge. The rock-hosted seafloor community was an order of magnitude more diverse compared to the seawater community directly above the rocks. With added H₂, peridotite-associated taxa increased assimilation of ¹³C-bicarbonate and ¹³C-acetate into 16S rRNA genes of operational taxonomic units by 146% (±29%) and 55% (±34%), respectively, which correlated with enrichment of H₂-oxidizing NiFe-hydrogenases encoded in peridotite-associated metagenomes. The effect of H₂ on anabolism was phylogenetically organized, with taxa affiliated with Atribacteria, Nitrospira, and Thaumarchaeota exhibiting the most significant increases in ¹³C-substrate assimilation in the presence of H₂. In SIP incubations with added H₂, an order of magnitude higher number of peridotite rock-associated taxa assimilated ¹³C-bicarbonate, ¹³C-acetate, and ¹³C-formate compared to taxa that were not associated with peridotites. Collectively, these findings indicate that the unique geochemical nature of the peridotite-hosted ecosystem has selected for H₂-metabolizing, rock-associated taxa that can increase anabolism under high H₂ concentrations. Because ultramafic rocks are widespread in slow-, and ultraslow-spreading oceanic lithosphere, continental margins, and subduction zones where H₂ is formed in copious amounts, the link between H₂ and carbon assimilation demonstrated here may be widespread within these geological settings.Subject terms: Microbial ecology, Water microbiology, Microbial biooceanography, Microbiome, Biogeochemistry     

Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy

Autophagy is a catabolic process essential for cell homeostasis, at the core of which is the formation of double-membrane organelles called autophagosomes. Atg9 is the only known transmembrane protein required for autophagy and is proposed to deliver membrane to the preautophagosome structures and autophagosomes. We show here that mammalian Atg9 (mAtg9) is required for the formation of DFCP1-positive autophagosome precursors called phagophores. mAtg9 is recruited to phagophores independent of early autophagy proteins, such as ULK1 and WIPI2, but does not become a stable component of the autophagosome membrane. In fact, mAtg9-positive structures interact dynamically with phagophores and autophagosomes without being incorporated into them. The membrane compartment enriched in mAtg9 displays a unique sedimentation profile, which is unaltered upon starvation-induced autophagy. Correlative light electron microscopy reveals that mAtg9 is present on tubular-vesicular membranes emanating from vacuolar structures. We show that mAtg9 resides in a unique endosomal-like compartment and on endosomes, including recycling endosomes, where it interacts with the transferrin receptor. We propose that mAtg9 trafficking through multiple organelles, including recycling endosomes, is essential for the initiation and progression of autophagy; however, rather than acting as a structural component of the autophagosome, it is required for the expansion of the autophagosome precursor.     

Protistan community patterns within the brine and halocline of deep hypersaline anoxic basins in the eastern Mediterranean Sea

Environmental factors restrict the distribution of microbial eukaryotes but the exact boundaries for eukaryotic life are not known. Here, we examine protistan communities at the extremes of salinity and osmotic pressure, and report rich assemblages inhabiting Bannock and Discovery, two deep-sea superhaline anoxic basins in the Mediterranean. Using a rRNA-based approach, we detected 1,538 protistan rRNA gene sequences from water samples with total salinity ranging from 39 to 280 g/Kg, and obtained evidence that this DNA was endogenous to the extreme habitat sampled. Statistical analyses indicate that the discovered phylotypes represent only a fraction of species actually inhabiting both the brine and the brine-seawater interface, with as much as 82% of the actual richness missed by our survey. Jaccard indices (e.g., for a comparison of community membership) suggest that the brine/interface protistan communities are unique to Bannock and Discovery basins, and share little (0.8–2.8%) in species composition with overlying waters with typical marine salinity and oxygen tension. The protistan communities from the basins’ brine and brine/seawater interface appear to be particularly enriched with dinoflagellates, ciliates and other alveolates, as well as fungi, and are conspicuously poor in stramenopiles. The uniqueness and diversity of brine and brine-interface protistan communities make them promising targets for protistan discovery.     

Introductions of non-native fishes into a heavily modified river: rates,patterns and management issues in the Paranapanema River (Upper Paraná ecoregion,Brazil)

Understanding the pathways and impacts of non-native species is important for helping prevent new introductions and invasions. This is frequently challenging in regions where human activities continue to promote new introductions, such as in Brazil, where aquaculture and sport fishing are mainly dependent on non-native fishes. Here, the non-native fish diversity of the Paranapanema River basin of the Upper Paraná River ecoregion, Brazil, was quantified fully for the first time. This river has been subject to considerable alteration through hydroelectric dam construction and concomitant development of aquaculture and sport fishing. Through compilation of a non-native fish inventory by literature review, with complementary records from recent field studies, analyses were completed on the timings of introduction, and the taxonomy, origin and introduction vectors of the non-native fishes. A total of 47 non-native fishes are now present across the basin. Of these, 24 invaded from the Lower Paraná River following construction of Itaipu Dam that connected previously unconnected fish assemblages. Activities including fish stocking, aquaculture and sport angling continue to result in new introductions. Discounting Itaipu invasions, the introduction rate between 1950 and 2014 was approximately one new introduction every 3 years. Introduced fish were mainly of the Cichlidae and Characidae families; most species were from other South American ecoregions, but fishes of African, Asian, North American and Central American origin were also present. These introductions have substantially modified the river’s fish fauna; when coupled with altered lentic conditions caused by impoundment, this suggests that the river’s native fishes are increasingly threatened.     

Comparison of metalloproteinase protein and activity profiling

Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.     

Amino acid metabolism of preimplantation bovine embryos cultured with bovine serum albumin or polyvinyl alcohol

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media, which is commonly replaced with synthetic compounds, such as polyvinyl alcohol (PVA). This study compared the effect of BSA and PVA on the development, blastocyst cell number and amino acid metabolism of preimplantation bovine embryos in vitro. Embryos were produced by in vitro maturation and fertilization of immature oocytes from abattoir-derived ovaries. Zygotes were cultured in synthetic oviduct fluid with either 4 mg/ml BSA (SOFaaBSA) or 1 mg/ml PVA (SOFaaPVA) in microdrops with a mineral oil overlay at 39 degrees C under a 5% O2/5% CO2/90% N2 atmosphere. Blastocyst rate and cell numbers were determined after 123 h of culture. In parallel, single expanding blastocysts grown in either medium were incubated in microdrops for 12 h. Amino acid profile of spent drops was determined by high performance liquid chromatography. Replacing BSA with PVA depressed blastocyst rate and cell numbers, and led to quantitative and qualitative differences in amino acid appearance, disappearance and turnover. These differences could partly be due to an increase in free intracellular amino acid concentration in SOFaaBSA embryos derived from hydrolysis of endocytosed BSA, and argue against the inclusion of PVA in bovine embryo culture media.     

Phylogenetic group distribution among <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from rivers in São Paulo State,Brazil

The phylogenetic group distribution of Escherichia coli strains isolated from the Sorocaba and Jaguari Rivers located in the State of São Paulo, Brazil, is described. E. coli strains from group D were found in both rivers while one strain from group B2 was isolated from the Sorocaba river. These two groups often include strains that can cause extraintestinal diseases. Most of the strains analyzed were allocated into the phylogenetic groups A and B1, supporting the hypothesis that strains from these phylogenetic groups are more abundant in tropical areas. Though both rivers are located in urbanized and industrialized areas where the main source of water pollution is considered to derive from domestic sewage, our results suggest that the major sources of contamination in the sampling sites of both rivers might have originated from animals and not humans.