Risks of floating microplastic in the global ocean.

Despite the ubiquitous and persistent presence of microplastic (MP) in marine ecosystems, knowledge of its potential harmful ecological effects is low. In this work, we assessed the risk of floating MP (1 µm-5 mm) to marine ecosystems by comparing ambient concentrations in the global ocean with available ecotoxicity data. The integration of twenty-three species-specific effect threshold concentration data in a species sensitivity distribution yielded a median unacceptable level of 1.21 ∗ 105 MP m-³ (95% CI: 7.99 ∗ 103-1.49 ∗ 106 MP m-³). We found that in 2010 for 0.17% of the surface layer (0-5 m) of the global ocean a threatening risk would occur. By 2050 and 2100, this fraction increases to 0.52% and 1.62%, respectively, according to the worst-case predicted future plastic discharge into the ocean. Our results reveal a spatial and multidecadal variability of MP-related risk at the global ocean surface. For example, we have identified the Mediterranean Sea and the Yellow Sea as hotspots of marine microplastic risks already now and even more pronounced in future decades.

Monoculture-based consumer-resource models predict species dominance in mixed batch cultures of dinoflagellates.

Global change will disturb the frequency, scale and distribution of harmful algal blooms (HABs), but we are unable to predict future HABs due to our limited understanding of how physicochemical changes in the environment affect interspecific competition between dinoflagellates. Trait-based mechanistic modelling is an important tool to unravel and quantify various direct and indirect interactions between species. The present study explores whether MacArthur's consumer-resource model can be used as a viable base model to predict dinoflagellate growth in closed multispecies systems. To this end, two batch culture experiments (294 cultures in total) with monocultures and multispecies cultures of Alexandrium minutum, Prorocentrum lima, P. micans, Protoceratium reticulatum and Scrippsiella trochoidea were performed. Despite changes to the relative (different nitrate concentrations) and absolute nutrient availability (dilutions of L1 medium), P. micans outcompeted all other species in mixed cultures. Consumer-resource modelling parameterized using monoculture growth correctly predicted this species dominance (R² between 0.80 and 0.95). Parameter estimates revealed that P. micans had a faster uptake of nitrogen when compared to its competitors, but did not differ in resource efficiency and natural mortality rate. Yet, while the model accurately predicted community dynamics during the growth phase, it was not able to predict their dynamics beyond the point of quiescence. Consumer-resource modelling was shown to differentiate the roles of resource assimilation, resource efficiency, and natural mortality rates in batch culture experiments with minimal data requirements beyond common measurements. The results suggest that consumer-resource models provide a promising basis for trait-based modelling of interspecific competition between (harmful) algae.

Toxic dinoflagellates and Vibrio spp. act independently in bivalve larvae.

Harmful algal blooms (HABs) and marine pathogens - like Vibrio spp. - are increasingly common due to climate change. These stressors affect the growth, viability and development of bivalve larvae. Little is known, however, about the potential for interactions between these two concurrent stressors. While some mixed exposures have been performed with adult bivalves, no such work has been done with larvae which are generally more sensitive. This study examines whether dinoflagellates and bacteria may interactively affect the viability and immunological resilience of blue mussel Mytilus edulis larvae. Embryos were exposed to environmentally relevant concentrations (100, 500, 2500 & 12,500 cells ml(-1)) of a dinoflagellate (Alexandrium minutum, Alexandrium ostenfeldii, Karenia mikimotoi, Protoceratium reticulatum, Prorocentrum cordatum, P. lima or P. micans), a known pathogen (Vibrio coralliilyticus/neptunius-like isolate or Vibrio splendidus; 10(5) CFU ml(-1)), or both. After five days of exposure, significant (p < 0.05) adverse effects on larval viability and larval development were found for all dinoflagellates (except P. cordatum) and V. splendidus. Yet, despite the individual effect of each stressor, no significant interactions were found between the pathogens and harmful algae. The larval viability and the phenoloxidase innate immune system responded independently to each stressor. This independence may be related to a differential timing of the effects of HABs and pathogens.

Common European harmful algal blooms affect the viability and innate immune responses of Mytilus edulis larvae.

Like marine diseases, harmful algal blooms (HABs) are globally increasing in frequency, severity and geographical scale. As a result, bivalves will have to face the combined threat of toxic algae and marine pathogens more frequently in the (near) future. These stressors combined may further affect the recruitment of ecologically and economically important bivalve species as HABs can affect the growth, viability and development of their larvae. To date, little is known on the specific effects of HABs on the innate immune system of bivalve larvae. This study therefore investigates whether two common harmful algae can influence the larval viability, development and immunological resilience of the blue mussel Mytilus edulis. Embryos of this model organism were exposed (48 h) to five densities of Pseudo-nitzschia multiseries or Prorocentrum lima cells. In addition, the effect of six concentrations of their respective toxins: domoic acid (DA) and okadaic acid (OA) were assessed. OA was found to significantly reduce larval protein phosphatase activity (p < 0.001) and larval viability (p < 0.01) at concentrations as low as 37.8 µg l(-1). P. multiseries (1400 cells ml(-1)), P. lima (150 cells ml(-1)) and DA (dosed five times higher than typical environmental conditions i.e. 623.2 µg l(-1)) increased the phenoloxidase (PO) innate immune activity of the mussel larvae. These results suggest that the innate immune response of even the earliest life stages of bivalves is susceptible to the presence of HABs.

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.

Contaminants within bacterial plasmid preparations trigger apoptosis in liposome transfected OVCAR3, but not in SKOV3 or AZ224 human ovarian cancer cells.

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.

Characterization of permanent cell lines that contain the AAV2 rep-cap genes on an Epstein-Barr-virus-based episomal plasmid.

Recombinant adeno-associated virus (rAAV) has emerged as a promising gene therapy vector. Its development, however, has been hampered by the lack of a readily available efficient production method. We investigated the possibility of establishing permanent cell lines for the production of rAAV with a new Epstein-Barr-virus (EBV)-based episomal AAV rep-cap plasmid (pCEP-rep/cap). HeLa and 293 cells were stably transfected with plasmids that carry the AAV2 rep/cap genes under transcriptional control of their endogenous promoters (p5, p19 and p40) either on the pCEP-rep/cap or an integrated (pIM45) plasmid. For the ease of monitoring transgene expression in live cells, a rAAV vector expressing gfp (the green fluorescent protein gene, rAAV-gfp/neo) was used. Establishment of stable transfected cell lines with these plasmids proved feasible but their usefulness was limited because of their instability. Within 8-12 weeks after their establishment, stably transfected rep-cap cell lines invariably lost their function. In addition, the rAAV-gfp/neo vector we used was susceptible to mutation in stably transfected HeLa cells. Our observations demonstrate specific problems both at the level of rep/cap gene function and the rAAV genome that can occur with the establishment of rAAV production cell lines. These experiments should aid the further development of efficient rAAV production protocols.

Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects.

The introduction of intracytoplasmic sperm injection (ICSI) has raised concern about safety in terms of a possible increase in the incidence of major congenital malformations, chromosomal aberrations or developmental problems. The possible influence of genetic imprinting on an ICSI procedure has not yet been investigated. We therefore studied the DNA-methylation status at a defined region in chromosome 15q11-q13 in 92 children born after an ICSI procedure. Imprinting defects in this region are associated with neurogenetic disorders, e.g. Angelman syndrome (AS) and Prader-Willi syndrome (PWS). Blood samples were taken directly after birth and stored at -80 degrees C. Genomic DNA purification was performed from 3-7 ml EDTA-blood. Sodium bisulphite treatment was carried out in order to distinguish methylated from unmethylated DNA by transferring the unmethylated nucleic acid cytosine into uracil and leaving the methylated cytosine unchanged. Subsequently, a methylation-specific polymerase chain reaction (M-PCR) was performed. In all 92 children (83 from ICSI with ejaculated spermatozoa and nine from ICSI with non-ejaculated spermatozoa), a regular DNA-methylation pattern was found in the PWS/AS region. In none of the children were clinical symptoms of PWS or AS present. In conclusion, the results of this study do not indicate a higher risk of DNA-methylation defects in children born after ICSI.

Clinical application of preimplantation genetic diagnosis for cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The gene was cloned in 1989 and the most frequent mutation was shown to be the delta F508 mutation. During PGD, embryos obtained in vitro are checked for the presence or absence of the mutation, after which only embryos shown to be free of the mutation are returned to the mother. Up to 1999, 48 intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) cycles had been carried out for PGD for CF in 24 couples, and different diagnostic tests had been used to select non-affected embryos. Thirteen patients became pregnant and 12 healthy babies have been born.

Chromosomal latency and expression at map unit 96 of a wild-type plus adeno-associated virus (AAV)/Neo vector and identification of p81, a new AAV transcriptional promoter.

OBJECTIVE: Human adeno-associated virus (AAV) is ubiquitous and known to establish latency by chromosomal integration. We have constructed a wild-type plus AAV vector, ins96-0.9Neo, containing the neomycin resistance gene open reading frame (Neo ORF) of 960 bases in length at map unit 96 of the virus. Ins96-0.9Neo was constructed in an unconventional manner in that the Neo ORF lacked a dedicated heterologous promoter. In this study, this wild-type plus AAV vector was to used to test AAV's packaging capacity and ability for chromosome 19 AAVS1 integration. However, when it was discovered that ins96-0.9Neo also transduced cells to G418 resistance, we also investigated the mechanism of Neo ORF expression in this vector. STUDY DESIGN/METHODS: We investigated the ability of ins96-0.9Neo to produce virus at high titers and to retain the Neo sequences by Southern blot analysis. The ability of ins96 0.9Neo virus to transduce the Neo gene into cells was analyzed by colony formation under G418 selection, and the ability of ins96-0.9Neo to latently infect cells, including the AAVS1 region of chromosome 19, was investigated by a series of polymerase chain reaction (PCR) amplifications. Finally, the RNA expression of the Neo gene at map unit 96 was investigated by reverse transcriptase primer extension (RTPE) analyses with two different primers and by S1 nuclease protection. RESULTS: High titers of the ins96-0.9Neo virus could be generated (10(9) infectious units [IU]/mL without concentration), the Neo gene was retained in the encapsidated viral genome, infection by this virus resulted in G418 resistance, and significant integration was taking place within the AAVS1 sequences of human chromosome 19 on transduction. Analysis of mRNA by RTPE using both primers and by the S1 nuclease protection assay mapped the 5' end of the Neo transcripts to approximately 700 bases upstream of the Neo ATG at map unit 81 (nt 3793-3813), thus identifying a new AAV promoter. CONCLUSIONS: These data demonstrate that ins96-0.9Neo will be useful for studying wild-type AAV integration and suggest that such wild-type plus recombinant AAV vectors may be useful for human gene therapy. The advantages of using such wild-type plus AAV vectors over defective AAV vectors include the ease in production of recombinant virus and the ability for site-specific integration into chromosome 19. This study also uncovered a previously unknown AAV promoter, p81. This finding suggests that the as yet uncharacterized ORF (nt 3922-4388) located just downstream of this promoter is likely an expressed gene. Furthermore, these data support our earlier findings that the AAV virion can package >900 bases more than can the wild-type.

The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis.

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.

Further definition of the sequence and position requirements of the arginine control element that mediates repression and induction by arginine in Saccharomyces cerevisiae.

Repression or induction of the genes involved in arginine biosynthesis or catabolism, respectively, both require participation of the ArgRp/Mcm1p regulatory complex. Our previous work showed that those opposite effects were mediated by a similar arginine-responsive element of 23 nucleotides (that we now call ARC, for ARginine Control) situated close to the start of transcription in the repressed promoters and far upstream of the TATA-element in the induced promoters. To define more precisely the sequence and position requirements of the ARC element, we have now characterized by mutagenesis the promoter elements of the arginine-repressible ARG1 and ARG8 genes. We also identify a functional ARC in the CPA1 promoter, thereby confirming, in agreement with our previous mRNA pulse-labelling data, the participation of a transcriptional component in the arginine regulation of that gene otherwise submitted to a translational regulation. From the 12 ARC elements now characterized, we have derived a consensus sequence and show that such a synthetic element is able to mediate ArgRp/Mcm1p-dependent arginine regulation. An important new finding illustrated by ARG1 and CPA1, is that contrary to what all the previous data suggested, repression can be mediated by ARC elements located far upstream of the TATA-box. The new data suggest that the arginine repressor might inhibit transcription in an active process.

Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine.

We have determined the sequences and positions of the cis elements required for proper functioning of the ARG3 promoter and proper arginine-specific control. A TATA box located 100 nucleotides upstream of the transcription start was shown to be essential for ARG3 transcription. Two sequences involved in normal arginine-mediated repression lie immediately downstream of the TATA box: an essential one (arginine box 1 [AB1]) and a secondary one (arginine box 2 [AB2]). AB1 was defined by saturation mutagenesis and is an asymmetrical sequence. A stringently required CGPu motif in AB1 is conserved in all known target sites of C6 zinc cluster DNA-binding proteins, leading us to propose that AB1 is the binding site of ARGRII, another member of the C6 family. The palindromic AB2 sequence is suggested, on the basis of published data, to be the binding site of ARGRI, possibly in heterodimerization with MCM1. AB2 and AB1 correspond respectively to the 5' and 3' halves of two adjacent similar sequences of 29 bp that appear to constitute tandem operators. Indeed, mutations increasing the similarity of the other halves with AB1 and AB2 cause hyperrepression. To mediate repression, the operator must be located close to the transcription initiation region. It remains functional if the TATA box is moved downstream of it but becomes inoperative in repression when displaced to a far-upstream position where it mediates an arginine and ARGR-dependent induction of gene expression. The ability of the ARG3 operator to act either as an operator or as an upstream activator sequence, depending on its location, and the functional organization of the anabolic and catabolic arginine genes suggest a simple model for arginine regulation in which an activator complex can turn into a repressor when able to interfere sterically with the process of transcription initiation.