Plastic litter is accumulating in ecosystems worldwide. Rivers are a major source of plastic litter to oceans. However, rivers also retain and transform plastic pollution. While methods for ...calculating particle transport dynamics in rivers are well established, they are infrequently used to quantify the transport and retention of microplastics (i.e., particles < 5 mm) in flowing waters. Measurements of microplastic movement in rivers are needed for a greater understanding of the fate of plastic litter at watershed and global scales, and to inform pollution prevention strategies. Our objectives were to (1) quantify the abundance of microplastics within different river habitats and (2) adapt organic matter “spiraling” metrics to measure microplastic transport concurrent with fine particulate organic matter (FPOM). We quantified microplastic and FPOM abundance across urban river habitats (i.e., surface water, water column, benthos), and calculated downstream particle velocity, index of retention, turnover rate, and spiraling length for both particle types. Microplastic standing stock was assessed using a habitat-specific approach, and estimates were scaled up to encompass the study reach. Spatial distribution of particles demonstrated that microplastics and FPOM were retained together, likely by hydrodynamic forces that facilitate particle sinking or resuspension. Microplastic particles had a higher downstream particle velocity and lower index of retention relative to FPOM, suggesting that microplastics were retained to a lesser degree than FPOM in the study reaches. Microplastics also showed lower turnover rates and longer spiraling lengths relative to FPOM, attributed to the slow rates of plastic degradation. Thus, rivers are less retentive of microplastics than FPOM, although both particles are retained in similar locations. Because microplastics are resistant to degradation, individual particles can be transported longer distances prior to mineralization than FPOM, making it likely that microplastic particles will encounter larger bodies of water and interact with various aquatic biota in the process. These empirical assessments of particle transport will be valuable for understanding the fate and transformation of microplastic particles in freshwater resources and ultimately contribute to the refinement of global plastic budgets.
We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site‐specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs ...to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the “common deletion” (CD), a 4977‐bp repeat‐flanked deletion associated with adult‐onset chronic progressive external ophthalmoplegia and, less frequently, Kearns‐Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild‐type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof‐of‐principle that, through heteroplasmy manipulation, delivery of site‐specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species.
Synopsis
Mutations and rearrangements of mitochondrial DNA (mtDNA) are a common cause of human disease, where they often co‐exist with wild‐type mtDNA within a single cell. Mitochondrially targeted engineered zinc finger nucleases (mtZFNs) can phenotypically rescue a severe mtDNA‐mediated dysfunction and show future therapeutic potential.
Previously reported mtZFN constructs were redesigned, greatly improving target specificity and allowing their safe use in human mitochondria.
The capacity of the novel mtZFN design was validated by selectively degrading point mutant mtDNA associated with neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) and maternally inherited Leigh syndrome (MILS).
The use of the novel mtZFNs was expanded by selectively degrading mtDNA harbouring a pathogenic large‐scale deletion associated with adult‐onset chronic progressive external ophthalmoplegia (CPEO) and, less frequently, Kearns‐Sayre syndrome (KSS) and Pearson's marrow pancreas syndrome.
Data are provided demonstrating that elimination of deleted, pathogenic mtDNA molecules by mtZFNs is sufficient for full recovery of oxidative phosphorylation in a disease model cell line.
Mutations and rearrangements of mitochondrial DNA (mtDNA) are a common cause of human disease, where they often co‐exist with wild‐type mtDNA within a single cell. Mitochondrially targeted engineered zinc finger nucleases (mtZFNs) can phenotypically rescue a severe mtDNA‐mediated dysfunction and show future therapeutic potential.
The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) ...designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
Anthropogenic litter (i.e., trash, AL) on beaches has negative ecological and economic impacts. Beach AL is likely moved together with coarse particulate organic matter (CPOM, algae, leaves), but no ...previous studies have assessed AL and CPOM co-distribution. We measured AL and CPOM on four urban beaches in Chicago, Illinois, USA, along two transect types (pier-adjacent, non-pier adjacent) in which each has three habitats (upland, beach, strand line). As expected, AL and CPOM density were positively related across all transects. AL and CPOM were significantly higher adjacent to piers, and variable among habitats. Wood, leaves, and smoking-related AL were most abundant in beach and upland zones while glass and algal detritus were abundant at the strandline. Overall, AL and CPOM show ‘patchy’ distribution attributed to wind and wave movement toward accumulation sites. Beach CPOM is a hot spot of biological activity (e.g., microbes, invertebrates, birds). Therefore, mixing of AL and CPOM suggests organisms may be more likely to encounter AL in mixed accumulations. Efforts to reduce the abundance and biological impacts of beach AL will benefit from emphasizing organic matter accumulation sites.
Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base ...specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes ...can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that ...in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.
The ecology of anthropogenic litter (AL) (i.e., trash) in marine ecosystems is a growing field of research. Freshwater and marine ecosystems have similar AL densities, but research on the assemblage ...and accumulation rates of AL in freshwater environments is less common. We studied AL accumulation at Pratt beach, located on Lake Michigan in Chicago, IL, USA. The beach has a concrete pier at the south end, with two areas of grass bisected by a walking path. Pratt beach receives many daily visitors, but unlike other urban beaches, it has no daily municipal beach grooming. We established five 60 m transects parallel to the shoreline, with increasing distance to the shoreline. Each transect was divided into four habitat zones (i.e., pier, south vegetation, path, and north vegetation). All AL within 1 m of each transect was collected biweekly from March‐November 2015. AL density (No. m−2) and input rate (No. m−2 day−1) were highest at the pier, regardless of distance from the shoreline. Fall had the highest AL density relative to spring or summer. We concluded AL inputs at the pier were dominated by direct littering and the retention of wind‐ and wave‐blown AL at the pier's wall. The study beach had much higher AL than adjacent beaches which received municipal AL cleaning, suggesting cleaning is effective at reducing AL density. This study suggests that, given the mobile nature of AL by natural processes, infrequent sampling may underestimate total AL abundance. Management efforts for AL should be directed towards the greatest AL accumulation sites within a beach, and at prevention of direct littering from visitors.
Mouse zygote morphokinetics were measured during interphase, the mitotic period, cytokinesis, and two-cell stage. Sequences of rounder-distorted-rounder shapes were revealed, as were changing ...patterns of cross section area. A calcium chelator and an actin-disrupting agent inhibited the area changes that occurred between pronuclear envelope breakdown and cytokinesis. During cell division, two vortices developed in each nascent cell and they rotated in opposite directions at each end of the cell, a pattern that sometimes persisted for up to 10 h. Exchange with the environment may have been promoted by these shape and area cycles and persisting circulation in the cytoplasm may have a similar function between a cell's interior and periphery. Some of these movements were sporadically also seen in human zygotes with abnormal numbers of pronuclei and the two-cell stages that developed from these compromised human zygotes.
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